New Biotechnology

Bacterial microcompartments (MCPs) are versatile proteinaceous organelles that compartmentalize metabolic pathways, offering promising scaffolds for synthetic biology and metabolic engineering. However, designing customized nanobioreactors requires distinguishing structurally indispensable shell proteins from those that can be modified or deleted to tune shell permeability without disrupting core organelle assembly. In this study, we performed a systematic biophysical and metabolic characterization of the hexameric shell protein PduU to evaluate its potential as a modular platform for synthetic organelle engineering. We tested whether deleting pduU or selectively truncating its N-terminal {beta}-barrel domain preserves shell assembly, metabolite flux, and intermediate confinement. Our results demonstrate that PduU modifications alter shell permeability while fully maintaining organelle structural integrity, monodispersity, and electrostatic colloidal stability. Crucially, this modulation in permeability redirects internal metabolic flux toward the energy-generating propionate pathway, resulting in elevated cell biomass and significantly increased yields of propionate, an economically vital industrial platform chemical. By establishing that PduU is a non-essential structural component whose modification tunes small-molecule flux, this work highlights PduU as a flexible locus for shell engineering, providing a scalable strategy for biomanufacturing of high-value bio-based products in tailor-made MCP nanobioreactors.

Influenza A virus (IAV) defective interfering particles (DIPs) are regarded as promising broad-spectrum antivirals due to their high efficacy demonstrated in animal trials. Previously, an OP7 chimera DIP was generated that showed markedly enhanced antiviral efficacy compared to conventional IAV DIPs. In addition, a high-yield cell culture-based production process for OP7 chimera DIPs was established in perfusion mode. However, due to the technical complexity of this technology, we aimed to combine the advantages of perfusion in terms of yield with that of a simple and less complex batch process. In the present work, we established a fed-batch production process for OP7 chimera DIPs that allows a more straightforward implementation into existing production facilities. Using a feeding regime that was able to overcome the cell density effect, we achieved productive virus infection in stirred tank reactors until a cell density of 10E+6 cells/mL. Furthermore, we obtained a high virus titer of 3.8 log10(HAU/100 uL), corresponding to a 3.4-fold increase compared to a reference batch production. Moreover, the produced material demonstrated a significantly increased interfering bioactivity shown by an in vitro potency assay. Interestingly, this efficacy was similarly high as compared to OP7 chimera DIP material previously produced in perfusion mode, despite twofold lower virus titers. Finally, we demonstrate robustness of OP7 chimera DIP seed virus trains, which is significant step towards later pharmaceutical production. Further, corresponding GMP MDCK-PB2(sus) cell and OP7 chimera virus banks are now available for later biomanufacturing further use. In conclusion, the established fed-batch process offers a valid n additional option for pharmaceutical grade OP7 chimera DIP GMP manufacturing production under GMP conditions required for subsequent clinical testing and for later large-scale manufacturing.

In liquid fermentation of filamentous fungi such as Aspergillus oryzae, increased broth viscosity and biomass adhesion to bioreactor surfaces remain major challenges. We previously developed a hyphal dispersion mutant lacking two hyphal adhesion factors, namely cell wall -1,3-glucan (AG) and biofilm galactosaminogalactan (GAG) (AG{Delta}-GAG{Delta} strain). The culture broth of the AG{Delta}-GAG{Delta} strain has low viscosity, which improves mixing and enzyme production. However, mycelia still extensively attach to bioreactor walls and downstream equipment, which impairs mixing and reduces product recovery. The hydrophobin RolA, a surface-active protein of A. oryzae, densely coats conidia and hyphae and contributes to cell surface hydrophobicity. In this study, we disrupted the rolA gene in AG{Delta}-GAG{Delta} (AG{Delta}-GAG{Delta}-{Delta}rolA strain) and evaluated the effects of this disruption on hyphal adhesion to the walls of culture vessels, enzyme production, and bioreactor performance. At the flask scale, the adhesion to glass surfaces was significantly reduced and recombinant enzyme activity was increased by 10%. Improved culture recovery at the end of fermentation further increased total enzyme yield. In a lab-scale stirred-tank bioreactor, both growth and enzyme production were increased. Scaling-up to a 200-L bioreactor showed reduced agitation power consumption while improving hydrodynamic properties. Fermentation of AG{Delta}-GAG{Delta}-{Delta}rolA was successfully scaled up to a 3000-L bioreactor; consistent enzyme activity and improved flow circulation in the bioreactors were confirmed by computational fluid dynamics analysis. Overall, the AG{Delta}-GAG{Delta}-{Delta}rolA strain has increased enzyme production and scalability, supporting its suitability for industrial applications.

The objective of this study was to enhance the production of monensin and its derivatives in Streptomyces sp. ATCC 15413. To this end the contributions of medium composition and enzyme engineering on polyketide biosynthesis were assessed. Enzyme engineering was implemented through a single-point mutation in KS5 of the polyketide synthase (PKS). This mutation increased premonensin productivity up to 29-fold, revealing and alleviating a rate-limiting step in the multi-enzyme biosynthetic pathway. Medium optimization proved comparably effective, raising titers by at least an order of magnitude across strains. Moreover, medium optimization and ketosynthase mutagenesis acted additively in the premonensin strain, further boosting its production. Overall, our findings show that medium optimization is the dominant factor in maximizing monensin yields, while enzyme engineering can deliver targeted benefits in specific contexts.

Background: Hydroxyectoine is a high-value compatible solute with applications in cosmetics, healthcare, and biotechnology. Its microbial production has traditionally relied on halophilic organisms requiring high-salinity cultivation, motivating the development of non-halophilic production hosts. Corynebacterium glutamicum is an attractive platform because of its industrial robustness and high metabolic capacity through the aspartate-family amino acid pathway. Results: A synthetic hydroxyectoine pathway was constructed by combinatorially varying the predicted translation initiation rates of four translational units comprising ask, ectAB, ectC, and ectD. A library of C. glutamicum transformants was screened at 40{degrees}C using hydroxyectoine-associated thermoprotection as a functional selection principle. Fast-growing variants generally displayed increased hydroxyectoine formation and higher hydroxyectoine-to-ectoine ratios than a strain carrying the native pathway configuration. The selected combination reached a hydroxyectoine-to-ectoine ratio of 4.7 and was transferred into the lysine-producing strain DM1729SL in order to increase precursor availability and hydroxyectoine production. Carbon-limited fed-batch cultivations showed that both temperature and dissolved oxygen influenced production. The best performance was obtained at 30{degrees}C and 50% relative dissolved oxygen, yielding 12.9 g/L hydroxyectoine with a yield of 0.161 g/g glucose and a volumetric productivity of 0.26 g/L/h after glucose depletion. A subsequent hypoosmotic downshock increased extracellular hydroxyectoine recovery to 14.5 g/L. Conclusions: Thermal stress-based combinatorial pathway balancing enabled efficient de novo hydroxyectoine production in C. glutamicum. Integration of precursor-enhanced host metabolism, controlled oxygen supply, fed-batch cultivation, and osmotic downshock resulted in a substantial improvement over previously reported de novo hydroxyectoine production in this host and establishes C. glutamicum as a promising platform for further hydroxyectoine process development.

The trend towards concentrated animal feeding operations (CAFOs) has served to concentrate not only livestock animals but the waste they produce to comparatively smaller areas. The point-source nature of this waste is an opportunity for the recovery and valorization of the nitrogen therein. Such a process would be viable on small to intermediate scales and require minimal inputs at the CAFO. In this study, we demonstrate the potential of the biopolymer cyanophycin to serve as a medium for manure-nitrogen recovery. In the first step, genetically modified strains of Escherichia coli produce intracellular cyanophycin from mock manure hydrolysates. Next, cyanophycin is recovered from microbial biomass via acid solubilization and base precipitation using electrochemically generated acids and bases. Finally, to improve both the yield and recoverable fraction of cyanophycin produced, we leverage the tunability of our genetically engineered system to probe the impacts of cyanophycin synthetase solubility, N-domain activity, and cyanophycin molecular weight on cyanophycin recoverability. Collectively, this work serves as a proof of concept for nitrogen recovery from agricultural waste, aligning with global sustainability initiatives.

The increasing world population necessitates new sustainable nutrient sources, making microalgae like Chlorella sorokiniana interesting due to its rich nutrient profile and sustainable cultivation methods. With genetic optimization tools like CRISPR/Cas9, microalgae as a nutrient source can be improved even further. However, degradation of the rigid cell wall of microalgae, and thereby developing protoplasts, is often necessary prior to transformation, but monitoring protoplast development in spherical, single-celled organisms like C. sorokiniana is challenging using bright-field microscopy. Carbotrace 480 and 630 were tested as fluorescent markers of the cell wall of a C. sorokiniana mutant for protoplast detection, and Carbotrace 480 was successfully used to distinguish protoplast from normal cells in a cell suspension. The enzymes Driselase, Glucanex, Snailase, and Saczyme were tested in different combinations to degrade the cell wall of the mutant, with Snailase as the most effective yielding ~60 % protoplasts. This study provides a quick and easy tool for monitoring protoplast development in the microalgae C. sorokiniana, the first step to improve C. sorokiniana as a sustainable nutrient source using genetic optimization tools like CRISPR/Cas9.

L-Threonine is an essential amino acid widely used in the feed, food, and pharmaceutical industries, and microbial fermentation is now the dominant route for its production. Further increases in producer titers depend on random mutagenesis coupled with high-throughput screening (HTS), but progress has been limited by the lack of a biosensor with a broad operating range, high sensitivity, and target specificity. Here, we show that L-threonine induces the tryptophan-repressed trp promoter of Escherichia coli, a response that, to our knowledge, has not been described previously. Deletion analysis showed that both the trpO operator and the trpL attenuation element contribute to induction, and that the lowest basal expression occurs only when both elements are present. By optimizing the ribosome-binding site, the promoter -10 region, and TrpR expression, we constructed an L-threonine biosensor on a pACYC backbone. The sensor responded linearly to L-threonine from 0.1 to 30 g/L (R2 = 0.9895), with an induction ratio of up to 51.7-fold. Among the 20 proteinogenic amino acids tested, only L-threonine produced a detectable response. The biosensor distinguished strains with different L-threonine production capacities and guided the directed evolution of homoserine kinase (ThrB) at a conserved motif, yielding variants with brighter reporter fluorescence and increased L-threonine production. This work provides a high-performance biosensor based on native E. coli regulatory elements for screening L-threonine overproducers.

Myoglobin, an oxygen-binding animal protein, was engineered in Pisum sativum (pea) to explore its potential as a food ingredient and balance the amino acid profile. In this study, minimal expression cassettes and binary vectors were used to express bovine myoglobin using particle gun and Agrobacterium-mediated transformation, respectively. Successful integration and expression of the myoglobin gene was achieved in P. sativum, with both methods yielding similar transformation efficiencies (~1%). Expression analysis of T2 seeds revealed that Agrobacterium-mediated transformation-derived transgenic lines that expressed myoglobin under the regulation of a Soybean 7S seed-specific promoter and Tobacco Etch Virus (TEV) translation enhancer and a chimeric Rb7MAR Terminator (Ps-BpRG13 events) consistently yielded the highest level of expression (0.32-1.57% of TSP), while transgenic lines with myoglobin expression under the regulation of a Soybean Phaseolin promoter and Rb7MAR Terminator (Ps-BpRG14 events) resulted in moderate levels of heterologous protein expression (0.13-0.83% TSP). Transgenic events with constitutive 2xCaMV35S promoter, TEV translation enhancer and Rb7MAR terminator (Ps-BpRG15 events) exhibited the lowest level of myoglobin expression (0.09-0.14% TSP). Co-bombardment of two minimal expression cassettes - one with myoglobin under the regulation of the Phaseolin promoter and Rb7MAR Terminator and the other with the nptII selectable marker under the regulation of a 2X constitutive CaMV35S promoter, TEV translational enhancer and TNOS Terminator, yielded lines that exhibited variable expression (0.03-0.77% TSP), with some events comparable in expression to Agrobacterium-derived Ps-pRG14 events. To the best of our knowledge, this is the first report of producing a heme-containing animal protein, myoglobin, in peas, with potential implications for sustainable production of food ingredients and nutritionally fortified and value-added plant products using molecular farming.

Protein engineering and precision fermentation provide an opportunity to increase the value of food proteins by improving their solubility, stability, functionality, or nutritional composition. Here, we use {beta}-lactoglobulin ({beta}LG) as a model protein to investigate how state-of-the-art computational protein design approaches affect these properties. First, the deep learning-based design tool ProteinMPNN was used to alter up to 20% of {beta}LG residues for increased stability. Second, the physics-based modeling platform PyRosetta was used to find positions in {beta}LG accommodating increased branched-chain amino acid (BCAA) content and up to 10 residues were simultaneously exchanged. Experimental characterisation of ProteinMPNN and stabilised BCAA-enriched variants showed similar secondary structure and oligomeric state as native {beta}LG. ProteinMPNN variants gave increased titers and increased thermal stability up to 15 {degrees}C, and this correlated with changes in the rate of surface pressure in droplet tensiometry. Stabilized BCAA-enriched mutants had altered acid solubility. Correlations between computationally derived biophysical metrics and experimental properties are presented and suggest some predictive power for surface hydrophobicity on protein yield.

Valerolactam is a promising bio-based monomer for polyamide synthesis, but microbial production remains limited by inefficient 5-aminovalerate (5AVA) cyclization, by-product formation, and insufficient process optimization. In this study, Corynebacterium glutamicum was engineered for valerolactam production by expression of davBA from Pseudomonas putida and lysP from Escherichia coli, followed by conversion of 5AVA to valerolactam using the recently identified avaC gene from Collinsella intestinalis. Flask cultivations confirmed efficient valerolactam formation with only minor accumulation of the by-products L-lysine, 5AVA, and glutarate. Batch bioreactor experiments showed that higher glucose concentrations increased titers but also promoted by-product accumulation, whereas increasing the dissolved oxygen setpoint from 30% to 50% improved growth and volumetric productivity. Intracellular cofactor analysis revealed declining energy status and shifts in redox balance during production. Based on these findings, carbon-limited fed-batch cultivation at 50% rDO improved production performance, reaching 3.6 g/L valerolactam with a yield of 0.231 g/g and a volumetric productivity of 0.075 g/L/h, while minimizing by-product formation. These results establish AvaC-based C. glutamicum as a promising platform for sustainable valerolactam biosynthesis.

Ensuring the safety and suitability of proteins considered for food and biotechnology applications is an important challenge. Computational approaches can facilitate the prioritisation of candidate proteins for further investigation, particularly given the rapid growth of genomic and metagenomic sequence datasets. In this study, we developed computational models to distinguish edible plant tissue-associated (EPTA) proteins from non-EPTA proteins. The positive class comprised 939 proteins with experimentally validated evidence of expression in plant tissues commonly consumed by humans, hereafter referred to as EPTA proteins. The negative class comprised 939 proteins annotated in UniProt as toxic, allergenic, or antinutritional, hereafter referred to as non-EPTA proteins. Models were evaluated using five-fold cross-validation with a cluster-based partitioning strategy, ensuring that no protein in the validation set shared more than 40% sequence identity with proteins in the corresponding training set. First, composition-based sequence features were evaluated to establish a baseline for EPTA or non-EPTA classification, with the Extra Trees (ET) classifier achieving a validation AUROC of 0.848. To incorporate evolutionary information beyond amino acid composition, PSSM profiles were subsequently evaluated, yielding an AUROC of 0.842 with an SVM classifier. Finally, to capture richer sequence patterns and contextual information using protein language modelling, we evaluated ESM2-based models. The fine-tuned ESM2-T33 model achieved the highest validation AUROC of 0.925 and was selected as the final predictive model. The resulting approach was implemented as the EdiProPred web server (https://webs.iiitd.edu.in/raghava/edipropred/) and standalone software for computational classification of plant proteins. The EdiProPred framework provides a useful computational tool for prioritising candidate plant proteins and facilitating the exploration of novel protein resources for food and biotechnology applications.

Polyhydroxyalkanoates (PHAs) are microbial polyesters that represent a sustainable alternative to fossil fuel-based plastics. These biodegradable polymers have properties similar to conventional plastics and can be produced from waste materials. The key enzyme in PHA synthesis is PHA synthase (PhaC) and despite its importance, only a few PhaCs from extremophilic microorganisms have been characterised in detail. Here we characterised PHA synthase enzyme from the thermophilic bacterium Caldimonas thermodepolymerans DSM15344, an attractive candidate for PHA production from lignocellulosic residues due to its unique properties such as utilisation of xylose-rich substrates and high PHA yields on this pentose sugar. PhaC was successfully expressed in the surrogate host E. coli BL21 (DE3) GOLD, purified to homogeneity and its enzymatic activity was confirmed in vitro. The optimal pH range and temperature were determined as 6.3-6.9 and 50 {degrees}C, respectively. The melting temperature of PhaC was detected at 57.83 {+/-} 0.04 {degrees}C and the enzyme storage stability was assessed. The tertiary structure, including the enzyme active site, was predicted using available bioinformatic tools and the predicted catalytic residues were supported by site-directed mutagenesis. This study represents the first biochemical characterisation of PHA synthase from C. thermodepolymerans, expanding the limited set of characterised thermophilic PhaC enzymes.

With the rapid development of modern biotechnology, DNA vectors have become fundamental tools for inserting, transferring, and expressing specific gene sequences in various fields such as gene cloning, gene expression, gene editing, and gene therapy. However, when dealing with complex structured DNA sequences, traditional vector construction methods face challenges with low connection efficiency. This study proposes a new method for constructing recombinant vectors by employing a strategy of high-temperature treatment followed immediately by placement on ice, effectively reducing the complexity of DNA structures and enhancing the efficiency of PCR product-vector connection, thereby improving the construction efficiency of recombinant vectors. This paper describes the technical details of the method, experimental validation, and applications in gene cloning, gene recombination editing, and the preparation of gene therapy drugs, providing a new efficient tool for molecular biology experiments.

Obtaining sufficient amounts of pure protein for downstream applications such as native mass spectrometry (nMS) is often challenging, especially when expression yields are low or proteins are unstable. In these cases, the commonly required buffer-exchange step is a major bottleneck, as it often leads to substantial protein loss and compromises biophysical characterization. These challenges are exacerbated in insect or eukaryotic expression systems, where protein yields are typically lower than in bacteria, making protein loss during purification particularly detrimental. Standard lysis and purification buffers contain non-volatile components such as Tris, phosphate, HEPES and sodium chloride, which form adducts during electrospray ionization (ESI) interfering with the signal and therefore must be re-moved prior to nMS. To address protein loss associated with this mandatory buffer-exchange, we evaluated an affinity-purification workflow, in which non-volatile salts are excluded throughout purification and proteins are directly eluted into nMS-compatible ammonium acetate-based buffers. This approach eliminates the need for a separate buffer exchange step and enables rapid nMS analysis immediately after affinity purification. We show that common eluents used in His- and Strep- based affinity purification, such as imidazole, biotin, and desthiobiotin, are well tolerated at relevant concentrations, allowing acquisition of high-quality spectra suitable for determining protein stoichiometry and for monitoring enzymatic or assembly processes. Together, this fast-track affinity workflow increases protein recovery, shortens sample preparation and complements online exchange protocols, which are less suited for monitoring processes. It hence expands the applicability of nMS to proteins and protein complexes that are difficult to obtain in sufficient quantity using conventional purification and buffer exchange strategies.

mRNA vaccines constitute a promising new platform for infectious disease prevention, having demonstrated their efficacy in response to the COVID-19 pandemic. While mRNA attributes such as purity, integrity, 5'-capping, polyA tail length, and nucleobase lipidation are critical to vaccine efficacy, another key attribute of any vaccine mRNA is its translatability - that is, how much antigen protein is produced by ribosomal translation. To assess mRNA translatability, cell-free translation (CFT) can be employed. Advantages of CFT include its rapid readout (~5 hours), minimal sample consumption, and measurement of antigen translation directly from mRNA, thereby eliminating any variability associated with cell transfection efficiency. This is in contrast to cell-based methods, which require larger amounts of mRNA material and formulation in lipid nanoparticles or lipofectamine to facilitate cell transfection. When developing a CFT method to assay mRNA translatability, one key consideration is which CFT system to use as there are several commercially available systems from a variety of different organisms and cell types. Presented here is the evaluation of three eukaryotic CFT systems - wheat germ extract (WGE), rabbit reticulocyte lysate (RRL), and HeLa cell lysate (HCL) - for the purpose of developing an analytical method to assay the translatability of mRNA. The dynamic range, linear range, sensitivity to thermal stress, and sensitivity to potential impurities (e.g., EDTA, double-stranded RNA) were determined for each system. Although HCL translated less protein than either WGE or RRL systems, it showed several advantages, notably a larger linear range and greater sensitivity to mRNA thermal stress and dsRNA impurities. Therefore, while it is still worthwhile to screen different CFT systems, the results presented here suggest that HCL should be strongly considered when developing an analytical method to assay translatability for mRNA vaccines.

The primary question in this work is whether a turbidity trace contains more than one resolved kinetic event before any rate or aggregate-mass information is supplied. A blind, three-gate hierarchical Gompertz procedure applied to human interleukin-1 receptor antagonist (IL-1ra) at 53 {degrees}C in phosphate retained one phase at 1 and 2 mg/mL, but selected two phases at 5 and 10 mg/mL. Thus the first resolved multiphase behavior appears at the low protein concentration of 5 mg/mL. Source optical-rate markers were withheld until after selection. Those observed optical rates were then compared with blind-fit phase rates, and the same comparison was expressed on a conditional mass-equivalent scale using the published 5.3-fold Type-I/Type-II turbidity response ratio. A separate 50 {degrees}C citrate series (4, 6, 8, 10, and 14 mg/mL) also selected two phases in every retained trace; its printed Vmax values were used only as post-selection comparators. Finally, two challenging 40 {degrees}C high-concentration traces (180 and 200 mg/mL) were digitized from the instrument-output plot and each retained two phases. All three conditions are analyzed independently. Blind recognition is therefore the central result; the mass-equivalent conversion is a secondary, post-selection interpretation of optical phase rates. The absolute {micro}M/min scale remains conditional on an endpoint aggregate fraction measured elsewhere.

Rapid point-of-care (POC) diagnostics require sensitive, stable, and cost-effective reporter systems. While lateral flow assays (LFAs) are widely used, their sensitivity is often limited by the use of colloidal gold, whereas enzymatic amplification using horseradish peroxidase (HRP) is constrained by the difficulties of recombinant production in bacterial hosts. In this study, we present a protein engineering framework utilizing APEX2, a robust engineered peroxidase, as a next-generation signaling element. We optimized the expression of APEX2-His6 in E. coli, achieving high yields (64 mg/L) of a functional apoenzyme that was efficiently reconstituted in vitro (RZ value: 2.1). Furthermore, we investigated the development of a bifunctional reporter by genetically fusing APEX2 to a single-domain antibody (sdAb). While the production of the fusion protein encountered significant solubility challenges in E. coli (yielding ~0.5 microgram/liter culture), our results confirm that APEX2 retains high catalytic activity and stability when integrated into an LFA format. Using West Nile Virus (WNV) as a model system, we demonstrate that APEX2-mediated enzymatic amplification provides a clear colorimetric signal on nitrocellulose membranes. This work establishes APEX2 as a commercially viable, recombinantly accessible alternative to traditional peroxidases, providing a versatile platform for the development of high-sensitivity diagnostic tools.

Methyl 12-aminododecanoate (ADAME) is a key precursor for Nylon 12 synthesis and an attractive target for sustainable microbial production. However, efficient biosynthesis of ADAME requires the coordinated oxidation and transamination of methyl dodecanoate (DAME), and identifying compatible enzymes for this multistep pathway remains challenging. In this study, we applied a machine learning-based bioinformatic screening approach to identify alternative enzymes for DAME-to-ADAME bioconversion. Among 18 selected distantly related AlkB homologs, AlkBGp01 from Alcanivorax sp. P2S70 showed activity toward DAME, producing 12-hydroxydodecanoic acid methyl ester (HDAME) and 12-oxododecanoic acid methyl ester (ODAME) despite low sequence identity to Pseudomonas putida AlkB. Further screening identified compatible redox partners, AlkG60 from the same species as AlkBGp01 and AlkTp04, whose combination with AlkBGp01 resulted in almost sixfold higher ODAME production than that achieved with P. putida AlkBFGJLT. ODAME production was further increased by more than threefold by conjugating these three newly identified proteins. The optimized single-plasmid system comprising conjugated AlkBGp01, AlkG60, and AlkTp04, together with AlaD and newly identified omega-transaminases EAV41574 produced 0.28 mM gDCW-1 ADAME, representing a 5.6-fold improvement over the previously reported P. putida AlkBFGJLT-based system with AlaD-CV2025. These results demonstrate that machine learning-guided enzyme discovery can identify functional distantly related homologs and compatible enzyme combinations for constructing efficient synthetic pathways. This study also highlights the importance of optimizing pathway architecture, including enzyme conjugation and plasmid configuration, for improving microbial production of bio-based monomer precursors.

Multi-stage continuous bioprocessing can increase volumetric productivity, operational consistency, and process throughput, but its design is complicated by coupling among dilution rate, reactor volume, feed allocation, and cellular physiology. Here, we present ContiDesigner, available at https://chemnettools.anc.univie.ac.at/ContiDesigner/, a mechanistic steady-state framework and interactive web tool for the system-level design of continuous fermentation cascades. Comparing one- and two-stage configurations at equal total reactor volume and outlet flow, ContiDesigner reveals how internal flow and reactor volume allocation shape space-time yield and identifies productivity-maximizing operating conditions. Compared with one-stage processes, two-stage cascades favor lower over-all dilution rates, thereby preserving residence time in the production stage. The first-stage dilution rate approaches the corresponding one-stage productivity optimum, but the cascade optimum occurs earlier, reflecting a system-level compromise between biomass generation and production-stage residence time. However, two-stage operation outperforms optimized one-stage operation only when non-growth-associated production in the second stage is sufficiently strong, whereas increasing growth coupling favors one-stage operation. Two case studies demonstrate both the potential and limits of process intensification. An optimized two-stage design is predicted to achieve a more than 1.5 fold increase in space-time yield for poly-R-3-hydroxybutyrate (PHB) production compared with a published experimental five-stage cascade, whereas the lactic acid case study identifies conditions under which staging offers no advantage. ContiDesigner translates these design principles into an accessible workflow to explore feasible operating regions and prioritize cascade designs for experimental evaluation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/743657v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@ef58faorg.highwire.dtl.DTLVardef@1ba88a4org.highwire.dtl.DTLVardef@160edd3org.highwire.dtl.DTLVardef@9dda34_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIContiDesigner enables system-level design of continuous fermentation cascades C_LIO_LIHigh stage-one dilution supports biomass generation C_LIO_LILow stage-two dilution preserves productive residence time C_LIO_LIYet two-stage cascades favor lower overall dilution than one-stage systems C_LIO_LITwo-stage advantage requires strong non-growth-associated production in stage two C_LI