Synechococcus elongatus PCC 7942 is a widely used model cyanobacterium for diverse research fields and for bioproduction applications owing to its ease of cultivation, genetic modification, and ability to fix CO2. We found that a laboratory strain of S. elongatus PCC 7942 expressing the ydeH gene from E. coli exhibited pronounced biofilm formation and cell aggregation upon ydeH induction, despite the species rarely showing these phenotypes under standard laboratory conditions. Our findings demonstrate that this strain harbors latent potential for biofilm formation and aggregation through c-di-GMP synthesis, suggesting possible applications in biomass recovery. Further, we identified that xcsA2 (Synpcc7942_2151), a putative cellulose synthase gene containing a PilZ domain that binds c-di-GMP is an indispensable gene in this biofilm-producing phenotype and experimentally confirmed ydeH-induced cellulose-like β-1,4-linked glucans production. Notably, xcsA2 represents the first putative functional cellulose synthase gene involved in biofilm/aggregate formation in S. elongatus and suggests that this organism may serve as a useful platform for bacterial cellulose bioproduction. Overall, discovery of xcsA2 provides a new genetic basis for exploring cellulose synthase evolution in cyanobacteria and specifically opens possibilities for engineering cellulose bioproduction utilizing S. elongatus.

Optimizing catholyte pH is pivotal for intensifying H2-mediated microbial electrosynthesis (MES) of acetate from CO2 because pH co-governs gas-liquid transfer, cathodic overpotential, and acetogen's activity, yet their coupled effects on reactor performance remain poorly resolved. Here, we systematically investigated acetate production from CO2 across pH 7.0-9.0 by integrating H2 transfer, cell voltage, productivity, and microbial community shifts. Increasing pH enhanced H2 availability through higher mass-transfer rates, but also increased cathodic overpotential and cell voltage, establishing a trade-off between substrate supply and electrical energy demand. The optimum pH of 7.5 delivered the shortest lag phase (2.2 ± 0.3 d), the highest acetate titer (18.43 ± 0.92 g·L[-1]), near-complete H2 uptake, and peak coulombic efficiency up to 79%, yielding minimal electricity intensity (~26.95 kWh·kg[-1]). Further pH elevation to 8.5-9.0 disrupted this balance, leading to reduced productivity and sharply increased energy consumption (136.77-236.96 kWh·kg[-1]). Notably, the specific NaOH addition for pH control at pH 7.5 remained comparable to that at pH 7.0 due to greater productivity. Microbial community analysis revealed acetogen enrichment near pH 7.5 and diversification at pH 9.0. These results define a pH-centered design strategy that aligns H2 transfer with microbial kinetics while minimizing energy and base usage, offering guidance for process intensification and scale-up.

Long-duration human missions to Mars will require autonomous systems capable of converting in situ resources into structural materials, tools, and functional components. More broadly, such systems represent a class of resource-limited bioprocesses relevant to extreme-environment manufacturing. Here, we investigate synthetic autotrophic-heterotrophic consortia, inspired by lichen biology, as a platform for autonomous biofabrication from granular feedstocks. We experimentally screened filamentous fungi and paired them with diazotrophic cyanobacteria to identify mutually supportive consortia capable of sustained growth and biomineral production in the presence of Martian regolith simulant as the primary inorganic substrate, without external organic carbon or nitrogen inputs. Selected co-cultures exhibited evidence of metabolic coupling, and untargeted metabolomic analysis revealed coordinated reprogramming consistent with integrated carbon and nitrogen metabolism within the consortia. These systems facilitated mineral consolidation of regolith particles, demonstrating the feasibility of near-closed-loop biomineral production under resource-limited conditions. While integration with additive manufacturing remains conceptual, this study establishes a framework for engineering self-sustaining microbial consortia for biomaterials production and highlights opportunities for coupling metabolism with material synthesis in both extraterrestrial and terrestrial environments.

Lactonic sophorolipids (LSLs) are high-value glycolipid biosurfactants with superior bioactivities, yet their industrial application is hampered by the low lactonization efficiency of wild-type strains. In this study, we employed a transcriptomics-driven approach to decode the regulatory landscape of the underexplored yeast Starmerella riodocensis GT-SL1R and engineer it for high-titer LSL production. Comparative transcriptome analysis under nitrogen limitation revealed a massive metabolic reprogramming, identifying 497 upregulated genes, including the core SL biosynthetic cluster (CYP52M1, ugta1, ugtb1, at, and mdr1). Crucially, we identified a novel lactone esterase (SrLE) with only 49% similarity to the well-known SbLE from Starmrella bombicola. While growth-related pathways (ribosome biogenesis and DNA replication) were repressed, nitrogen recycling mechanisms were activated to fuel SL biosynthesis. Directed metabolic engineering of the lactonization pathway through SrLE or SbLE overexpression, coupled with fed-batch fermentation in a 5-L bioreactor, achieved exceptional LSL titers of 189.33 g·L[-1] and 191.45 g·L[-1], respectively. Conversely, SrLE knockout resulted in the exclusive production of acidic SLs, confirming its pivotal role in lactonization. These results provide deep mechanistic insights into SL biosynthesis and demonstrate the power of targeted enzyme engineering in creating robust microbial cell factories for specialized glycolipid production.

Continuous downstream processing of messenger ribonucleic acid (mRNA) therapeutics requires purification strategies that are scalable, robust, and capable of removing product- and process-related impurities from in vitro transcription mixtures. Precipitation-based purification coupled with tangential flow filtration (TFF) has recently been demonstrated as a chromatography-free route for continuous mRNA purification. However, implementation of this approach requires real-time monitoring strategies that can distinguish the product-rich precipitated phase from the impurity-rich liquid phase, as well as phase-dependent transport behavior across the integrated process. In this work, we develop a phase-resolved process analytical technology (PAT) and residence time distribution (RTD) framework for continuous mRNA purification by PEG/NaCl precipitation and sequential TFF. Inline UV-Vis spectroscopy and LED-based optical density measurements were used concurrently to separate nucleic-acid absorbance from turbidity-driven responses associated with suspended mRNA precipitates. RTD experiments with dissolved and precipitated tracers revealed phase-dependent transport behavior in both the tubular precipitator and TFF module, which was quantified using axial dispersion and tanks-in-series models. During integrated operation, inline PAT captured startup, steady-state, and washout behavior, while stable TMP indicated operation without observable membrane fouling. This work demonstrates that precipitation-based continuous mRNA purification can be strengthened through phase-resolved monitoring and RTD-based process understanding, providing a framework for robust development of continuous downstream processes for nucleic acid therapeutics.

Accurate prediction of bioprocess scale-up is critical for accelerating the deployment of novel and sustainable biomanufacturing systems. However, this remains challenging as multi-scale data is expensive to generate and mechanistic understanding is often incomplete, leading upscaling decisions to rely heavily on empirical expertise. This work proposes a data-efficient strategy that integrates hybrid modelling with transfer learning to construct a high-fidelity model from limited lab scale data and then adapt it using only pilot scale information for industrial scale prediction. A key innovation is the explicit assessment of this framework under small-data scenarios, reflecting the practical constraints of industrial development. Using a real-world yeast fermentation case, the proposed framework achieved accurate industrial scale dynamic predictions with a mean absolute percentage error of 23.2%, demonstrating its high data efficiency. Furthermore, this study reveals that the greyness of hybrid models exerts a decisive influence on its predictive accuracy and the feasibility of transfer learning under data scarcity, and that its optimal level differs from scenarios with abundant data. These findings therefore provide the first guidance on how to exploit hybrid modelling and transfer learning to build scalable digital twins when data are limited, enabling more confident and reliable bioprocess development and upscaling.

The biocatalytic asymmetric amination of bulky N-heterocyclic ketones is often limited by low enzyme activity and poor performance at high substrate loading. Herein, we developed an efficient (R)-selective transaminase for the synthesis of (R)-1-Boc-3-aminopiperidine through an integrated strategy combining phylogeny-guided enzyme mining, structure-guided engineering, and mechanistic analysis. Screening of transaminases identified MbATA as the "best" parent enzyme, affording the target amine from N-Boc-3-piperidone (1B3OP) in specific activities of 0.269 U·g[-1] and > 99% ee. Subsequent engineering yielded variant M3 (H61L/Q57R/P158Q), which exhibited a 45-fold increase in activity over the wild type (WT) while retaining strict enantioselectivity (> 99% ee). M3 achieved completely aminated 500 mM substrate loading within 4 h, 1000 mM in an extended duration of 24 h. Preparative biotransformation afforded 8.5 g and 15.6 g of product in 85% and 78% isolated yields, respectively. Structural and mechanistic analyses showed that the beneficial mutations enlarged and remodeled the substrate-binding pocket, strengthened favorable substrate interactions, shortened the reactive cofactor-substrate distance, and promoted productive binding conformations. These results establish M3 as a practical biocatalyst for high-substrate-loading synthesis of chiral piperidine amines.

Enzyme screening platforms are typically evaluated based on their throughput and analytical performance rather than their ability to mimic the conditions encountered at large scale. In bioreactors, enzymes experience gradients in substrate concentration, oxygen availability, pH, temperature, and interfacial exposure, caused by imperfect mixing and multiphase operation. These gradients affect both enzyme activity and stability, yet they are rarely incorporated into the assays used to select biocatalysts. Traditionally, a variant is chosen based on its performance under static, well-mixed laboratory conditions, yet those conditions do not necessarily represent the process conditions it must eventually perform under. Microfluidic devices can in principle recreate process-relevant conditions at a small scale since these devices offer precise control over the flow, residence time, and multiphase contact. Yet most current platforms are not built to mimic the scale-down process-derived conditions. Here, we assess current microfluidic tools against this process-relevant conditions and outline how modular designs can be combined into sequential screening platforms that generate the conditions in a bioreactor, for early-stage biocatalyst selection.

Hepatocytes are cells of the hepatic parenchyma that perform a series of essential functions, representing about 80% of its mass. Due to their importance, the use of hepatocytes has gained prominence in scientific research, mainly in the prediction of hepatotoxic drugs. As primary hepatocyte culture presents limitations, human induced pluripotent stem cells (hiPSC) have been used as a promising alternative. However, the differences between 2D and 3D culture during the differentiation of iPSCs into hepatocytes is limitedly explored. In this context, this study compared the differentiation of hepatocytes-like (iHep) from hiPSC in two-dimensional (iHep-2D), three-dimensional (iHep-3D) and mixed (iHep 2D-3D) cultures to evaluate their impact on the acquisition of hepatocyte-like characteristics. After 2D differentiation, transcriptomic analysis revealed that iHep-2D has a gene profile very similar to that of primary human hepatocytes, demonstrating that this could be a valuable protocol to use as a starting point for iHep 2D-3D differentiation. The cells were also evaluated for the presence of hepatocyte-associated markers by immunofluorescence and our results showed that, although all three cultures generated iHep cells that were able to express specific hepatocyte markers, the iHep 2D-3D differentiation generated cells displaying a more pronounced hepatocyte-like phenotype, based on Albumin and CYP3A4 expression. Therefore, in this study we suggest that combining 2D and 3D differentiation may promote a more pronounced hepatocyte-like phenotype in iHeps derived from human iPSCs, which could serve as a valuable model to investigate liver disease.

Gibberellins (GAs) are ubiquitous phytohormones that regulate plant growth and are widely used in agriculture. Among these, GA3 and GA4+7 are the only commercially available products, yet GA4+7 commands a much higher price than GA3, primarily due to its low titer in the industrial fungus Fusarium fujikuroi. To engineer a high-yielding GA4+7 producer, we first deleted p450-3 to block the conversion of GA4 and GA7 to GA1 and GA3. This led to the accumulation of GA4+7 at 0.934 g/L, a 37-fold increase over the wild-type strain, albeit with over half reduction in total GA accumulation. Using this Δp450-3 mutant as a platform, we combined metabolic engineering (overexpressing rate-limiting enzymes) with process optimization (pH, medium composition and fermentation duration). These combinatorial interventions synergistically boosted GA4+7 production. Under optimized conditions, the engineered strain achieved a final titer of 6.08 g/L (a 6.51-fold increase over the Δp450-3 parent), comprising 2.13 g/L GA4 and 3.96 g/L GA7, representing 5.30- and 7.44-fold increases, respectively. Preliminary optimization in a 10 L fermenter yielded 3.51 g/L of GA4+7. Finally, a solid-state fermentation system was developed on wheat bran, yielding 17.34 g GA4+7 per kg and enabling green, in-house, in-situ gibberellin production. In addition to substantially increasing the GA4+7 titer and total GA accumulation, this study demonstrated that the GA4/GA7 ratio can be modulated through both molecular and fermentation strategies.

Vero cells remain an indispensable continuous substrate for human viral vaccine manufacturing. Despite decades of empirical process optimization, intrinsic genomic instability, including segmental aneuploidy and dynamic chromatin rearrangements, continues to limit the durability of engineered phenotypes under sustained viral burden and bioreactor stress. Here, we review the expanding engineering toolkit for the Vero lineage across a three-layered functional framework: the membrane interface, cytoplasmic foundry, and nuclear blueprint, evaluating translational prospects at each level. Receptor transplantation and morphological reprogramming have broadened viral entry range and enabled suspension-adapted culture formats, while metabolic flux management and temporally controlled apoptosis modulation have addressed intracellular production bottlenecks, albeit often with trade-offs between productivity, biosafety, and long-term population stability. At the genomic level, targeted perturbations of transcriptional regulators and emerging epigenetic interventions offer more durable gains, yet expression drift, clonal heterogeneity, and karyotypic instability during extended passaging highlight the need for locus-level precision rather than constitutive trait installation. Looking forward, infection-responsive dynamic logic circuits and the systematic identification of Vero-specific genomic safe harbors could shift the paradigm toward a conditionally responsive manufacturing architecture. Collectively, these advances suggest a pathway for transitioning the Vero lineage from a passive, empirically optimized biological substrate into a conditionally responsive, genomically stable, and programmable platform for modern vaccine preparedness.

Autonomous cell-based control of heterologous gene expression can simplify batch-culture bioprocessing by eliminating external monitoring and extrinsic control of culture conditions. Existing approaches use auto-induction media, synthetic cell-cell communication systems, or application-specific biosensors. A simpler, resource-efficient, and general-purpose expression control system responsive to common changes during batch culture would be highly valuable. We used native E. coli promoters, including PhdeA, PdpS, PfumA, PrpoA, PrpoS, PgadA, and PyiaG, and recombinase-based switches to repurpose endogenous transcription signals for control of heterologous gene expression. Specifically, natural changes, covering 1-2 orders of magnitude across growth phases, in transcription from endogenous promoters result in recombinase expression at the exponential-to-stationary phase transition. So-expressed recombinases invert a constitutive promoter regulating expression of arbitrary heterologous genes amplifying the endogenous transcriptional input signal by more than 10-fold. We realized reversible and single-use switching with reduced static and dynamic cell-to-cell variation and overall expression amplification. We used "off-the-shelf" genetic parts and abstraction-based composition frameworks to realize reliable forward engineering of our synthetic genetic systems. We engineered autonomous control systems for regulating heterologous gene expression. Our system uses generic endogenous promoters to sense and control heterologous expression during growth-phase transitions. Our system does not require specialized auto-induction media, production or activation of quorum sensing, or the development of application-specific biosensors. Cells programmed to control themselves could simplify existing bioprocess operations and enable the development of more powerful synthetic genetic systems. Modern biotechnology uses engineered microbes to manufacture molecules that are incorporated into medicines, flavors, fuels, and other materials. Making molecules inside cells requires enzymes, whose expression levels and timing need to be optimized to maximize final product yields. Many engineered microbes use enzymes that should only be expressed during the later stages of cell growth to avoid overconsumption of resources or accumulation of toxic intermediates. Here, we develop simple genetic devices that enable engineered cells to control themselves, automatically switching on or off enzyme expression during batch cell culture. Unlike existing approaches, our devices do not require specialized growth media or wasteful synthesis of cell-cell signaling molecule.

Peptide phage display is a cornerstone technology in molecular engineering, providing a robust platform for the rapid discovery of high-affinity ligands. The rapid rise of peptides, especially in therapeutics, over recent years has shed light on the importance of phage display as a tool for peptide discovery. The evolution of peptide scaffolds from flexible linear sequences to structurally constrained architectures has expanded the scope of application of peptides in general. The review examines strategies used to generate monocyclic, bicyclic, and multicyclic peptides. These constrained formats mimic the complex loops of natural proteins, offering enhanced metabolic stability and superior binding affinity for challenging targets like protein-protein interaction interfaces. Furthermore, the review surveys the diverse applications ranging from targeted therapeutics and molecular diagnostics to the engineering of material-specific peptides for nanotechnology. Finally, we discuss current technical hurdles and the transformative potential of integrating next-generation sequencing and computational modeling to redefine the future of peptide discovery.

Microbial siderophores are high-affinity iron-binding compounds which are produced by bacteria, fungi, and actinomycetes to obtain iron and survive and interact with different species in an iron-deficient environment. While the conventional research on siderophore systems deals mainly with the study within the same taxa, modern researchers have increased their inclination toward cross-kingdom integration of siderophore behavior and their impact on host-associated environments. This can be largely attributed to differences in biosynthetic gene clusters, receptor systems, and regulatory networks, which produce distinct genotype-to-phenotype results determining microbial cooperation and competition. Current advancements in genomic research, together with omics studies like transcriptomics, proteomics, and metabolomics, have created newer insights into how siderophores function. However, the present literature evidences multiple major gaps in multi-omics data because the link between genomes and metabolomes remains weak due to inconsistent regulatory data sets and failure in identifying producer-consumer relationships in polymicrobial systems. Additionally, major constraints like molecular instability, delivery system limitations, host toxicity, limitations in upscaling, and regulatory issues delimit the use of siderophores in medical treatment, agricultural practices, and environmental biotechnology. This review aims to bridge the existing knowledge about siderophore biochemistry, biosynthesis, ecological functions, and genetic regulation across kingdoms while integrating multi-omics outlook with translational considerations. Thus, by connecting molecular mechanisms with evolutionary cross-talk, this study aims to provide a system-level framework in the world of siderophore-mediated iron uptake and therefore shapes future directions in emerging fields of microbial engineering, precision therapies, and sustainable biotechnology.

28-epi-homobrassinolide (28-epi-HBL), a bioactive analog of the steroidal phytohormone brassinolide, exhibits broad biological activities and considerable application potential. However, conventional chemical routes remain limited by tetrahydroxylation step that relies on highly toxic osmium tetroxide in combination with costly chiral ligands, whereas the Baeyer-Villiger oxidation step requires strong oxidants and often exhibits limited regioselective control. In this work, optimization of tetrahydroxylation was achieved using the Shing-Plietker reaction, thereby avoiding the use of osmium tetroxide. Moreover, an engineered Baeyer-Villiger monooxygenase (CpnB_BL) was heterologously expressed and used for regioselective B-ring lactonization under mild conditions. A ZIF-8-based immobilization strategy was established for this engineered enzyme, further improving its stability and solvent tolerance, and increasing the isolated yield of the Baeyer-Villiger step to 83%. These optimized strategies were subsequently applied to the total synthesis of 28-epi-HBL, achieving an overall yield of 44.9%. This represents a marked improvement over the highest previously reported yield in the literature, which stands at approximately 20%. Collectively, this study lays the groundwork for the development of more precise and sustainable synthetic routes to 28-epi-HBL.