The antibiotic pipeline is in crisis, even though microbial natural products, account for most approved antibacterial drugs and retain vast, untapped biosynthetic potential. Genuinely new scaffolds are rare because discovery remains constrained to a narrow set of cultivable taxa and expressed biosynthetic gene clusters, while most microbial chemistry persists as "dark matter" in uncultured organisms and silent clusters. Each bottleneck is both a discovery and a biotechnology challenge: a predicted cluster must be expressed, its product isolated, and its biosynthesis engineered to workable titres before any hit can realistically progress. This review links the major obstacles in the contemporary pipeline including limited ecological sampling, silent and cryptic clusters, access to uncultured bacteria, inefficient metabolome mining, weak genome-metabolome integration, and incomplete target identification, to the technologies best suited to address them, drawing on recent case studies of integrated workflows rather than single-platform approaches. By evaluating these strategies for both discovery power and bioprocess tractability, we outline a practical framework for turning microbial dark matter into producible, mechanistically understood antibiotic leads.

Phase separation, which drives the formation of dynamic and reversible membraneless biomolecular condensates has emerged as a novel molecular strategy for adaptation to environmental stresses. In this review, (i), summarize how this process enables rapid and precise signal integration and regulation, (ii), integrate recent advances in the study of plant biomolecular condensates and (iii), systematically summarize the mechanisms of stress-induced phase separation. We discuss how plants utilize phase separation to orchestrate molecular responses to abiotic stresses, as well as biotic stresses such as pathogen invasion. Furthermore, we provide a focused analysis of the potential roles of endogenous plant molecules in modulating phase separation processes. Finally, we address the key questions and challenges in the field and propose a "plant condensatome" research roadmap integrating multi-omics approaches including genomics, transcriptomics, and proteomics to comprehensively map the composition, dynamic properties, and environmental responsiveness of plant condensates. We firmly believe that interdisciplinary integration to deeply resolve the mechanisms of phase separation will provide a new theoretical foundation for understanding plant environmental adaptation strategies and advancing molecular breeding for stress-resistant crops.

Methanotrophs are bacteria with the ability to directly utilize methane as substrate to produce different useful biomaterials (e.g., lipids, proteins, bioplastics, methanol). However, due to the solubility and mass transfer limitations of methane, the methanotroph biotechnology remains almost entirely in laboratory/research scale. To address this, studies in recent years have focused on increasing the methane volumetric mass transfer coefficient (kLa) through modifications of bioreactor design and operations. This review critically analyzed these advances, aiming to identify the most suitable strategy for methane bioconversion. The modifications were categorized, i.e., mechanical agitation, feed gas introduction system, and cultivation medium modification [including addition of a non-aqueous phase (NAP)], and their effectiveness was related to kLa and/or productivity metrics (PM) (i.e., yield, elimination capacity, and remediation efficiency). Studies that determined both kLa and PM were assessed using a new metric (∆PM/∆kLa, with ∆ as the difference between implemented strategy and control). This metric, which was independently determined for each study, allowed cross-study comparison of the effectiveness of the different modifications/strategies. A positively high ∆PM/∆kLa indicates greater emphasis on PM improvement (or biological performance) over kLa enhancement (or physical attributes) of the bioreactor system. While majority of strategies result in a positive ∆PM/∆kLa, two-phase partitioning bioreactors (TPPBs) exhibited the highest potential (i.e., highest ∆PM/∆kLa). The effectiveness of this strategy relies on the homogeneous dispersion of the added NAP. Although TPPB operation is more complex than conventional bioreactors, this review suggests that future research should consider TPPB strategies to address its complexity and economic viability.

Precise and scalable control of gene expression across biological contexts remains a fundamental challenge in synthetic biology and therapeutic development, and massively parallel reporter assays (MPRAs) have emerged as a transformative platform for systematically interrogating cis-regulatory elements (CREs) and enabling high-throughput and quantitative dissection of sequence-dependent transcriptional activity to address this challenge. Beyond their original role in empirical characterization, the convergence of barcoded MPRA technologies with deep learning-based modeling has substantially expanded their impact, facilitating predictive inference and the de novo design of synthetic regulatory elements with programmable behavior. In this review, we synthesize recent advances in barcoded MPRA platforms and AI-driven modeling frameworks for the rational engineering of CREs with tunable transcriptional strength and specificity. We further highlight cutting-edge applications in microbial synthetic biology, mRNA therapeutics, and cell-specific gene regulation, emphasizing the role of barcoded MPRA in supporting data-efficient and cross-context generalizable design. Finally, we discuss current technical and conceptual limitations and future directions toward safe, programmable, and clinically relevant regulatory control systems, outlining a roadmap for next-generation cis-regulatory engineering and gene therapies.

Succinic acid (SA) is a key C4 platform molecule with wide industrial relevance, including bio-based polymers and solvents. Current reviews largely emphasize traditional producers (e.g., Actinobacillus, Mannheimia, and Basfia) and rarely synthesize progress in Enterobacteriaceae. Here, we close this gap by providing a systems-level review of SA biomanufacturing in Enterobacteriaceae, focusing on non-model genera (Enterobacter, Klebsiella, Citrobacter) that combine fast growth, facultative anaerobiosis, broad feedstock utilization, and high engineering amenability. We outline the metabolic blueprint underpinning SA formation, highlighting the reductive TCA branch, anaplerotic CO2 fixation (PEPC/PEPCK), the glyoxylate shunt, and redox constraints that govern carbon partitioning. We then summarize recent advances in strain development, including pathway rewiring, stronger carboxylation, adaptive laboratory evolution, and dynamic control, that increase SA titers, yields, and productivities. Finally, we discuss downstream processing and techno-economic bottlenecks, biosafety and regulatory considerations, and emerging directions including division-of-labour consortia and circular-carbon integration for scalable, carbon-efficient SA biorefineries.

Fructosyltransferase (FTF), classified within glycoside hydrolase families GH32 and GH68 of the GH-J clan, plays an indispensable catalytic role in the biosynthesis of fructooligosaccharides (FOS) and fructan polymers (such as inulin and levan). These enzymes specifically catalyze fructosyl transfer reactions, constructing structurally diverse carbohydrates through β-(2 → 1) or β-(2 → 6) glycosidic linkages: FOS (linear oligosaccharides with DP 2-10 and a terminal glucose) and fructan polymers (high-molecular-weight polysaccharides). The resulting products not only exhibit excellent physicochemical properties but also demonstrate multiple physiological benefits including prebiotic activity, dietary fiber functionality, and regulation of glucose/lipid metabolism, showing broad application prospects in functional foods and pharmaceutical fields. This review systematically summarizes the microbial source diversity, three-dimensional structural characteristics, substrate recognition mechanisms and the molecular basis for the diversity of catalytic products of microbial FTF, including levansucrase, inulosucrase and β-fructofuranosidase. Focusing on enzyme molecular engineering strategies such as directed evolution, rational design, and semi-rational design, this review clarifies the intrinsic relationship between the catalytic properties of FTF and the structural and functional characteristics of their products, and discusses their application value in the synthesis of functional carbohydrates. This paper provides important theoretical support for the fundamental research of FTF and the development of precision synthesis technologies for FOS and fructans.

The European Union is currently engaged in an economic transition towards a circular bioeconomy that is expected to combine more sustainable growth with greater industrial sovereignty and resilience. Consequently, European competitiveness increasingly depends on the ability of advanced technologies, such as biotechnology, to provide the basis for manufacturing goods. These range from fuels and chemicals to biobased materials, advanced food ingredients, and a range of biopharmaceuticals. However, unlocking the potential of enabling technologies for the circular bioeconomy is a major undertaking. It involves harnessing the power of two game-changing technologies, biotechnology and advanced digital technologies (especially AI). To achieve this, it is imperative for Europe to bring together its knowledge, expertise, and infrastructure assets and to promote the concept of data federation. In this regard, Europe is fortunate to have European research infrastructures (RI) that provide ideal frameworks to achieve this. Here, a vision of how e-Infrastructures, such as the one embedded in the Infrastructure for Biotechnology Services for Biomanufacturing (IBISBA), can contribute to the digitalisation of biotechnology and catalyse the growth of biomanufacturing is presented. By advancing asset sharing, interoperability, federated modelling, and data-driven innovation, such RI can significantly accelerate both scientific discovery and industrial application.

Branched-chain amino acids (BCAAs) are recognized not only as industrially important fermentation products but also as versatile biosynthetic platforms for the production of branched-chain value-added chemicals. The characteristic branched carbon skeletons of BCAAs, together with their metabolically versatile intermediates, enable the biosynthesis of a broad spectrum of value-added chemicals, including branched-chain α-keto acids (BCKAs), alcohols, and carboxylic acids. This review systematically examines the metabolic basis of BCAA biosynthesis and the features underpinning its platform functionality, followed by a comprehensive overview of metabolic engineering strategies for constructing efficient BCAA-producing prokaryotic cell factories. Particular emphasis is placed on bacterial production platforms, especially Escherichia coli and Corynebacterium glutamicum, in which engineering carbon flux through enhanced glucose uptake, expanded pyruvate availability, reinforced biosynthetic flux, regulated cofactor supply, and improved product export. Advances in enzyme engineering are also discussed, with four complementary strategies highlighted for expanding the branched-chain chemical repertoire: enhancement of catalytic activity, improvement of substrate selectivity, engineering of cofactor utilization through NADH regulation, and optimization of enzyme expression. Finally, current challenges and future directions are discussed, and an engineering framework is proposed for the rational development of BCAA platform-based biomanufacturing toward an expanded repertoire of branched-chain chemicals.

The increasing accumulation of organic waste and the demand for renewable chemicals highlight the production of medium-chain fatty acids (MCFAs) as an effective strategy for waste valorization. Microbial chain elongation (CE) converts short-chain fatty acids (SCFAs) into MCFAs. However, its efficiency in mixed cultures is frequently limited by unstable carbon flow, restricted electron transfer, and shifts in functional microbial communities. While exogenous materials are increasingly applied to regulate CE, their mechanisms of action have not been as systematically reviewed as traditional process optimization strategies. This review examines how material-mediated CE enhances MCFA production by facilitating metabolic pathways, directing microbial enrichment, and promoting electron transfer. Specifically, the discussion focuses on carbon-based materials, iron-based materials, and engineered composites, detailing their role in electron shuttling, interfacial redox buffering, microbial attachment, and functional microbial community assembly. Furthermore, we summarize material design principles, their enhancement effects, and the key factors governing operational stability in practical mixed-culture systems. Rather than focusing solely on the enrichment of single functional microbes, this review emphasizes the application of exogenous materials to direct microbial community assembly and enhance interfacial microbe-material interactions. These mechanisms provide a basis for understanding and regulating caproate selectivity during organic waste conversion. Finally, by incorporating economic and environmental considerations, we propose strategies for scaling up material-assisted CE from the laboratory to practical applications. Ultimately, this review builds a framework connecting material properties, microbial ecology, and process efficiency, facilitating the selective conversion of organic waste into MCFAs.

Cell wall-degrading Carbohydrate-Active enZymes (CAZymes) have substantial potential for biological control of crop diseases by targeting the cell walls of filamentous plant pathogens (fungi and oomycetes) and could substitute for environmentally hazardous pesticides. The cell walls of fungi and oomycetes share common components but also differ in important ways, and there have been considerable recent advances in understanding dynamic structural differences across fungal species. The damage caused to cell walls by CAZymes and the potential of CAZymes in biocontrol are reviewed using studies with purified CAZymes and genetic studies in which CAZyme mutant strains have been tested for altered antagonism and biocontrol. The primary scope encompasses fungal mycoparasite CAZymes (e.g., β-1,3-glucanase, β-1,6-glucanase, α-1,3-glucanase, chitinase, cellulase, and carbohydrate-binding modules), and the secondary, more selective, scope includes bacterial and plant-derived CAZymes that offered novel insights into cell wall damage or facilitated comparisons with fungal-derived CAZymes. Mycoparasite regulation of cell wall-degrading CAZymes is complex, with few transcription factors and small-molecule inducers well understood. Plant pathogen responses to mycoparasite cell wall-degrading CAZymes involve compensatory changes mediated via cell wall integrity signaling. We discuss strategies to improve the effectiveness of CAZymes in antagonism and biocontrol, and ways to engineer a hyper-mycoparasite alongside risk assessments of adverse or unintended effects. These strategies partly depend on bridging knowledge gaps in the cell wall structures of oomycetes and fungal plant pathogens, and in how both mycoparasites and plant pathogens protect their own cell walls from the damaging effects of cell wall-degrading CAZymes.

The separate collection of bio-waste across the European Union has positioned anaerobic digestion (AD) as a key process, enabling simultaneous energy recovery through biogas production and organic recycling through digestate application to soils. However, plastic contamination in bio-waste streams, primarily from food packaging, challenges process performance, digestate quality, and overall circularity. To mitigate conventional plastic contamination, biodegradable plastics have been proposed as alternatives in bio-waste management, yet their behavior and fate under anaerobic conditions remain uncertain. This review presents a global state-of-the-art analysis of plastic contamination in bio-waste AD, with particular emphasis on the role of biodegradable plastics. It first situates AD within the conceptual and regulatory framework of bio-waste organic recycling and summarizes key bio-waste characteristics relevant to AD. Current knowledge on the anaerobic biodegradability of biodegradable polymers is then synthesized, including strategies proposed to enhance degradation, providing a basis for analyzing experimental studies on commercially available biodegradable plastic products with respect to biodegradability, process stability, energy performance, and microbial community dynamics. The review further examines plastic contamination in AD systems by synthesizing reported contamination levels in bio-waste streams and digestates for both macro- and microplastics derived from conventional and biodegradable polymers. Analytical methodologies used to characterize this contamination are critically assessed, with attention to their suitability and limitations for complex organic matrices and biodegradable plastics. Finally, the environmental implications of digestate land application are discussed, and key research perspectives are identified to clarify the role of biodegradable plastics and advance circularity in bio-waste AD systems.

Acyl-homoserine lactones (AHLs), the canonical quorum-sensing signals of Gram-negative bacteria, have attracted increasing attention as cross-kingdom regulatory molecules that influence microalgal growth, carbon allocation, redox homeostasis, extracellular organization, and algal-bacterial interactions. However, their potential as predictable regulators of microalgal biotechnology remains uncertain because of limited mechanistic understanding and substantial variability in reported responses. This review critically evaluates AHL chemistry, biosynthesis, environmental fate, quorum quenching, biological responses, and engineering implications, and integrates these aspects into an evidence-based framework for AHL-mediated microalgal regulation. Current evidence indicates that AHL structural features influence physicochemical properties and signal behavior, but a consistent structure-response relationship across microalgal species has not been established. Reported responses depend strongly on effective signal exposure, which is shaped by AHL stability and transformation, quorum-quenching activity, microbial context, cultivation conditions, and physiological state. At the cellular level, AHL exposure is associated with changes in photosynthesis, redox regulation, carbon partitioning, lipid metabolism, and population-level organization. The molecular mechanisms underlying AHL perception in microalgae remain experimentally unresolved. From an engineering perspective, AHL-mediated regulation represents an emerging approach to biological process regulation. Multi-omics, quantitative sensing, machine learning (ML), reactor-scale modelling, and controlled signal delivery may support mechanistic analysis, exposure monitoring, response prediction, and process optimization, although their application to AHL-mediated microalgal systems remains at an early translational stage. Future progress requires standardized AHL quantification, mechanistic validation, microbial-consortium engineering, and reactor-scale verification, together with integrated techno-economic and biosafety assessments to evaluate the reliability and scalability of AHL-mediated strategies for microalgal biotechnology.

Biomanufacturing has emerged as a strategic emerging industry worldwide, offering sustainable alternatives to conventional chemical manufacturing. To support this development, diverse production chassis have been developed, including microorganisms, mammalian cell culture systems, and plant-based platforms. Among these, plants represent a promising chassis for the biosynthesis of high-value products, as they directly fix CO2 through photosynthesis and possess sophisticated metabolic networks that facilitate the production of structurally complex molecules. Recent advances in synthetic biology have increased the programmability of plant systems and enabled their development as engineered production chassis for the production of high-value products. This review integrates recent advances in natural and synthetic genetic elements including promoters, terminators, and transcription factors (TFs) that facilitate chassis development are summarized. Recent advances in the elucidation, reconstruction, and optimization of biosynthetic pathways for representative classes of valuable compounds, including alkaloids, terpenoids, and phenylpropanoids, are then highlighted, with particular emphasis on pathway engineering strategies that enhance productivity. Finally, we outline the key challenges and future perspectives for the large-scale industrial applications of plant chassis. This review provides a comprehensive and timely perspective on plant-based biomanufacturing, offering conceptual guidance and practical insights for advancing plant chassis from laboratory research to sustainable industrial applications.

Sustainable agriculture requires innovative technologies that improve crop productivity while reducing environmental impacts. Electrospun nanofibers (ESN) have emerged as promising materials due to their high surface area, tunable structure, and ability to incorporate and release bioactive compounds in a controlled manner. This review examines ESN as an integrative approach for delivering bioactive substances and beneficial microorganisms directly to soil and plants. Recent advances in the production, evaluation, and agricultural applications of ESNs are discussed, with particular attention given to the use of biodegradable polymers and the integration of live microorganisms into nanofibrous matrices. Beyond their role as delivery platforms, ESN may interact with soil microbial communities through their surface properties, degradation behavior, and effects on local resource availability. However, direct evidence linking ESN application to changes in soil microbiome structure and function remains limited. Nevertheless, critical limitations-such as scalability, high production costs, sterility challenges, and potential ecological risks-must be addressed. A thorough understanding of ESN-microbiome-soil dynamics, supported by standardized characterization, long-term field studies, and molecular tools, is crucial for their safe and effective deployment in sustainable agriculture.

Branched-chain amino acids (BCAAs), comprising L-valine, L-leucine, and L-isoleucine, are essential amino acids with extensive applications in food, feed, pharmaceuticals, and cosmetics. Escherichia coli and Corynebacterium glutamicum, the two predominant industrial workhorses have been extensively engineered for high-level BCAAs biosynthesis. This review presents a comprehensive analysis of recent advances in microbial BCAAs production through a Mechanism-Module-Process framework. From the mechanism dimension, the intricate BCAA biosynthetic architectures are delineated, encompassing allosteric feedback inhibition, transcriptional attenuation, and transport system. From the module dimension, modular metabolic engineering strategies are dissected, including precursor supply enhancement, NADPH cofactor rebalancing, biosensor-driven dynamic regulation, and adaptive laboratory evolution for strain robustness. From the process dimension, how oxygen availability reprograms cellular metabolism across aerobic, microaerobic, and anaerobic fermentation is systematically examined, and how two-stage fermentation coupled with cofactor rebalancing resolves the growth-production trade-off to achieve near-theoretical yields is discussed. Furthermore, the biosynthetic strategies and current production status of BCAAs derivatives are summarized, highlighting the importance of multidimensional framework engineering. Collectively, this Mechanism-Module-Process framework provides a holistic roadmap for constructing superior BCAAs cell factories and extending their metabolic potential toward derivative biosynthesis.