Sulfolobus islandicus, an emerging archaeal model organism, offers unique advantages for metabolic engineering and synthetic biology applications owing to its ability to thrive in extreme environments. Although several genetic tools have been established for this organism, the lack of well-characterized chromosomal integration sites has limited its potential as a cellular factory. Here, we systematically identified and characterized 13 artificial CRISPR RNAs targeting eight integration sites in S. islandicus using the CRISPR-COPIES pipeline and a multi-omics-informed computational workflow. We leveraged the endogenous CRISPR-Cas system to integrate the reporter gene lacS and validated heterologous expression through a β-galactosidase assay, revealing significant positional effects. As a proof of concept, we utilized these sites to genetically manipulate lipid ether composition by overexpressing glycerol dibiphytanyl glycerol tetraether (GDGT) ring synthase B (GrsB). This study expands the genetic toolbox for S. islandicus and advances its potential as a robust platform for archaeal synthetic biology and industrial biotechnology.

Piezoelectric scaffolds are emerging as promising therapeutic strategies for musculoskeletal regeneration. These materials convert mechanical forces, ranging from intrinsic motion to focused ultrasound (US) force, into localized electrical cues for tissue-specific musculoskeletal regeneration. Mechanistically, piezoelectric-converted electrical energy activates mechanosensitive and voltage-gated channels that trigger early regenerative signaling pathways. In this review, we describe the fundamental principles of the piezoelectric material class that focus on dipole alignment, geometry, and activation paradigms to culminate in their differential effects on the regeneration of musculoskeletal tissues. We also discuss lead-free platforms, closed-loop systems, as well as printable constructs capable of delivering wire-free electrical stimulation (ES). Finally, we discuss current translational challenges and future directions and practical steps toward clinical adoption of piezoelectric scaffolds.

A biofoundry integrates laboratory automation with Design-Build-Test-Learn (DBTL) workflows to accelerate strain development for sustainable manufacturing. Quantifying the economic efficiency of automated processes remains challenging. Here, we define the robot-assisted module (RAM) as a plug-and-play unit for constructing workflow and apply the Experiment Price Index (EPI), a standardized metric that combines time and cost per sample to evaluate and optimize synthetic biology workflows. Using EPI calculation and RAMs, we developed four workflows for strain development: guide (g)RNA cloning, genome editing, DNA assembly, and sample analysis. EPI identified workflow bottlenecks, elimination of redundancies, and assessment of techno-economic tradeoffs. We further extended the EPI framework on techno-economic assessment (TEA) by estimating return on investment (ROI) and payback periods for biofoundry operations at varying project scales. Our results demonstrate EPI for cost-effective experimental planning and scalable biofoundry deployment. Beyond strain engineering, EPI serves as a universal tool for evaluating automation efficiency across biotechnology.

The COVID-19 pandemic highlighted the urgent need for advanced lung regenerative medicine. While traditional research focused on biochemical pathways, biophysical cues are equally critical regulators of lung cell behavior. This review discusses the role of key mechanical cues including cyclic stretch, strain/pressure, geometry, and matrix stiffness on lung cells in health and disease. The focus is on the evaluation of biomimetic platforms (decellularized scaffolds, dynamic surfaces, biomaterial constructs, and lung-on-chip devices) that recapitulate these environments; and the paradigm shifts in the field which show the importance of physiologically relevant systems. Finally, we identify challenges and future directions for translating mechanobiology-informed approaches into clinical therapies, highlighting their transformative potential for lung tissue engineering.

Unicellular algae are appealing for nutritional and biotechnological utility, but show wide variation across strains and can be challenging to produce. Species in the thermo-acidophilic genus Galdieria use diverse organic carbon sources for fermentative growth, which can include waste-stream feedstocks, while having complete amino acid compositions for human nutrition. Here, we investigated the metabolic dynamics of Galdieria to catalog organic carbon conversion to biomass. Tested strains had enhanced growth upon 3% CO supplementation, triggering efficient glucose uptake to reach ~5 ± 0.3 g dry biomass l. Stable-isotope analysis revealed that organic carbon uptake dominated CO fixation in darkness under mixotrophy, with CO an apparent metabolic trigger. Galdieria yellowstonensis 5587.1 was able to consume up to 8.3 g carbon l day from industrial confectionery waste, with C-phycocyanin (C-PC) reaching 3.8% of dry biomass and remaining thermostable at 72°C. Thus, this framework can be used to optimize Galdieria-based bioprocesses for the conversion of inexpensive waste into high-value biomass, and identifies CO as a trigger of organic carbon assimilation, even in heterotrophic conditions.

Antivenom is the only effective treatment for snakebites, but it often fails to tackle venom-induced local tissue/muscle damage, leading to permanent disabilities. We investigated whether monoclonal single-chain variable fragments (scFvs) targeting myotoxin II (M-II) from Bothrops asper venom, delivered via mRNA-lipid nanoparticles (LNPs), could neutralise M-II-induced adverse effects under diverse settings. Human cultured myotubes transfected with mRNA-LNPs expressed scFvs within 24 h and showed resistance to both M-II and venom. In a mouse model, a single intramuscular injection of mRNA-LNPs prompted scFv expression within 48 h and protected against M-II-induced damage. This approach reduced biomarkers for muscle injury, myonecrosis, and damage to the basement membrane and vasculature. This study demonstrates the use of mRNA technology in snakebite management, serving as a proof of concept to improve treatments for envenomings. Although a prophylactic approach may not be feasible for snakebites, prompt delivery of antibodies at the bite site might improve patient outcomes.

Kleptosomes - specialized organelles in sea slugs that hijack chloroplasts - illuminate pathways for synthetic organelle engineering. Their unique mechanisms of organelle integration, energy coupling, and stress-responsive adaptation offer potential blueprints for bioenergy and medical applications, merging fundamental research and cutting-edge biotechnology to address global sustainability and health challenges.

Due to health and pollution concerns, sustainable textile production is receiving increasing attention. Bacterial cellulose (BC) offers an environmentally friendly alternative to petroleum-based fabrics. However, the use of petroleum-based synthetic colorants and toxic reagents in dyeing processes impedes the fully sustainable production of colored BC. To address this issue, we developed a one-pot production system for colored BC biosynthesis, aiming to simplify and streamline the manufacturing process. A co-culture strategy using Escherichia coli for natural colorant synthesis and Komagataeibacter xylinus for BC synthesis was developed and optimized to achieve the one-pot production of multicolored BC, including proviolacein-BC (green), prodeoxyviolacein-BC (blue), violacein-BC (navy), deoxyviolacein-BC (purple), astaxanthin-BC (red), β-carotene-BC (orange), and zeaxanthin-BC (yellow). This study demonstrates the robust co-culture strategy and platform for efficient, scalable, and eco-friendly process biopolymer-based fabric production, offering a sustainable alternative for applications in the living materials industry.

In vivo chimeric antigen receptor (CAR) engineering has advanced into clinical trials with encouraging safety and efficacy. Technology breakthroughs, including targeted lipid nanoparticles, genetically modified lentiviruses, and optimized CAR constructs, have enabled precise, efficient, and safe gene delivery. This forum summarizes innovations accelerating the clinical translation of in vivo CAR engineering for immunotherapy.

Microbially induced biomineralization (MIBM) is a trending biotechnological application with the potential to be used in the engineering of built environment; however, it is limited by premature germination of spores due to stresses during concrete mixing. We investigated the germination dynamics of Paenibacillus alkaliterreae (PA) spores under variable temperature, pH, and salinity levels to identify the operating ranges conducive for spore germination and biomineralization in an outdoor environment. To prevent premature germination, the spores were covered with a protective coating of extracellular polymeric substance (EPS). The spores germinated and biomineralized CaCO across environmental conditions of 25-37°C, 0.5-4% NaCl, and pH 6-10. EPS-coated spores achieved 88 ± 2.8% self-healing in 28 days compared with 72 ± 2.7% in free spores. Chemical and morphological characterization confirmed that the bioconcrete comprised CaCO biomineral in the form calcite, which improved the strength, durability, and quality of this bioconcrete over conventional concrete.

The rise of complex biotherapeutics has introduced bottlenecks in production using mammalian cells. Clustered regularly interspaced short palindromic repeats (CRISPR)-based screens enable unbiased discovery of engineering targets that mitigate biomanufacturing-relevant constraints. This forum gives an overview of recent advances and remaining challenges in applying CRISPR screening to build robust, modality-specific cell factories.

Chronic wounds present a significant clinical challenge due to their complex pathophysiology and resistance to standard treatments. A key obstacle in developing therapies is the lack of animal models that accurately mimic human chronic wound characteristics. Existing rodent models fail to replicate critical features, such as delayed re-epithelialization and unresolved inflammation, while larger animals, including porcine models, also fall short. Here, we introduce a novel glutaraldehyde-induced porcine model that mimics key aspects of human chronic wounds. Glutaraldehyde causes dermal toxicity, resulting in impaired structural integrity, oxidative stress, persistent inflammation, and bacterial colonization. Analyses showed features such as delayed healing, extracellular matrix (ECM) disruption, mitochondrial dysfunction, and chronic inflammatory responses. Comparative transcriptomic and lipidomic studies revealed shared signaling pathways and metabolite profiles with human venous leg and diabetic foot ulcers, highlighting the translational relevance of the model. This innovative platform offers valuable insights into chronic wound mechanisms and aids the development of effective targeted therapies.

Extracellular vesicles (EVs) have gained significant attention as therapeutics, building from natural mechanisms of paracrine signaling. The field has evolved with substantial heterogeneity in methods to isolate and characterize EVs and new methods are needed to scale-up EV production for therapeutic use. In this study, we isolated EVs from four porcine donors of bone marrow-derived mesenchymal stromal cells (MSCs) via three different cell culture methods: standard tissue culture plates, a 3D printed perfusion bioreactor, and microcarriers in spinner flasks. We explored EV manufacturing yield, characteristics, and content via proteomics and RNAseq. The MSC donor and their cell culture method affected the yield of EVs produced, whereas the method of EV isolation dominated the clustering of protein and RNA contents. As a step towards therapeutic application, in vitro tubule formation and hypoxic cardiac spheroid contraction assays showed improvements in vasculogenesis and cardiac cell recovery, respectively, in the presence of EVs.

The emerging field of biosensors exploits the abilities of cells to identify specific molecules, presenting improved sensitivity, specificity, and limit of detection. Whole-cell biosensors (WCB) are organisms specifically engineered to detect a target analyte and express a reporter in response. In biomanufacturing, they can be used for monitoring of key substrate and metabolite concentrations or strain engineering, while in medicine, they can be used to diagnose disease or report on human-microbe interactions. Many applications require WCB to coexist with mammalian cells where a key challenge is to keep separate cell populations viable while still allowing them to interact. In this review, we highlight key considerations when encapsulating WCB to engineer controlled microenvironments that enable collaboration and coexistence of different populations.

Semiconductor biohybrid systems leverage microbial enzymatic precision and semiconductor light harvesting for efficient solar-to-chemical conversion, offering sustainable alternatives to energy-intensive production. To overcome challenges associated with the synthesis of ecofriendly nanoparticles (NPs), we utilized wastewater pollutants to produce semiconductor biohybrids. We engineered sulfate-reducing bacteria [rSRB-polyhydroxybutyrate (PHB)] for the self-assembly of extracellular polymeric substance (EPS)-complexed NPs through microbial S-metal bonding. These semiconductor biohybrids drive sulfate reduction using photogenerated electrons, while simultaneously degrading pollutants through hole-mediated oxidation, thus establishing a self-reinforcing cycle that enhances PHB synthesis. Photoelectrons fuel the acetogenic Wood-Ljungdahl pathway (WLP) through c-type cytochromes, enabling solar-driven CO fixation for acetyl-CoA and NADPH regeneration. Coupled photoredox reactions channel carbon flux into PHB biosynthesis, achieving a yield of 15.38 g/l. In addition, photoelectrons upregulate sulfate metabolism genes, stabilizing metal sulfide production. Thus, this system achieves solar-driven CO reduction coupled with organic conversion into chemicals in wastewater bioreactors, providing a sustainable route for pollutant removal and carbon mitigation, advancing low-carbon wastewater treatment and a circular bioeconomy.

Gene therapy has emerged as a promising strategy for tissue regeneration, offering the potential to address the limitations of conventional treatments. In this review we present an overview of applications of gene therapy in tissue regeneration, emphasizing recent advancements and future directions. Our work addresses gaps in the current literature by examining developments in molecular biology and genetics, such as clustered regularly interspaced short palindromic repeats (CRISPR) gene editing, advances in 3D bioprinting, and progress in gene delivery for tissue engineering. We describe case studies and clinical trials that demonstrate the potential of gene therapy applications in tissue engineering. We conclude by highlighting challenges and future directions, including emerging technologies and personalized gene-based approaches for tissue engineering research.

Mycoprotein (MP) production represents a promising environmentally sustainable strategy to address global protein deficit. To enhance the nutritional profile and production efficiency of MP, we employed CRISPR/Cas9-mediated scarless gene knockout and obtained a Fusarium venenatum strain (designated FCPD), which exhibited a 32.9% increase in essential amino acid index (EAAI) through targeted truncation of competitive metabolic pathways and regulation of amino acid metabolism or biosynthesis. FCPD achieved a 44.3% reduction in substrate consumption while improving MP production rate by 88.4% compared with the wild type (WT) strain. The cradle-to-gate life cycle assessment (LCA) shows that FCPD could reduce environmental impacts such as global warming potential (GWP) by 4-61.3% under production scenarios in six representative countries. Comparative environmental performance demonstrated the superiority of FCPD-MP over cell-cultured meat and chicken meat. These findings establish CRISPR/Cas technology and metabolic engineering as the dual-purpose tool for both nutritional enhancement and environmental impact mitigation in alternative protein production.

While regulatory functions of mature miRNAs are well established, the functions of miRNAs* and their potential for genetic engineering in crop improvement remain underexplored. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) to generate artificial miR2118a/b (amiR2118a/b) by editing miR2118a/b-5p and obtained several amir2118a/b mutants in soybean (Glycine max). miR2118a/b-5p modifications altered the secondary structure of precursor amiR2118a/b (pre-amiR2118a/b) and reduced mature miR2118a/b levels. These amir2118a/b mutants retained the ability to initiate biogenesis of phased small interfering RNAs (phasiRNAs), albeit with a reduced abundance compared with wild-type (WT) plants. Furthermore, these mutants upregulated the expression of genes related to growth and defense under normal and Pseudomonas syringae pv. glycinea (Psg)-infected conditions, respectively. Notably, two transgene-free amir2118 mutants exhibited enhanced resistance to Psg, soybean cyst nematode (SCN), and root-knot nematode (RKN), and achieved increased yield under pathogen-free field conditions. This study provides a strategy to generate artificial miRNAs (amiRNAs) for crop improvement through the CRISPR/Cas system by mutating miRNAs* in crops.

Methane (CH) represents an abundant source of carbon and energy with significant potential for sustainable biotechnological processes. For efficient bioconversion of CH into value-added products, synthetic methanotrophy has emerged as a promising strategy, enabling the rational design of engineered microbial systems. In this review, we highlight recent advances in CH-based bioprocesses, covering the metabolic design of synthetic methanotrophs and optimization of bioreactor systems adapted for gas fermentation. A comparative analysis of key CH-converting enzymes is provided, with particular emphasis on soluble methane monooxygenase and its heterologous production in industrial chassis. Recent progress in modular one-carbon (C1)-pathway engineering accelerates enzyme optimization and highlights synthetic methylotrophy as a prerequisite for robust synthetic methanotrophy. Collectively, these advances establish a foundation for scalable, efficient, and sustainable CH-based biotechnological processes.

Chromoproteins (homologous to fluorescent proteins identified initially in marine organisms) are visible to the naked eye under white light. Their unique advantages - including robust expression in both prokaryotic and eukaryotic systems, broad color diversity, and tunability through genetic engineering - make them desirable tools for synthetic biology. This opinion article provides a comprehensive overview of recent progress in chromoprotein-based research, highlighting their physicochemical properties, detailed classification, engineering strategies, and emerging applications. We anticipate that ongoing discovery and rational design of chromoprotein variants will significantly advance the development of visible chromoprotein reporters, enabling more accessible and modular platforms for synthetic biology research and applications.