Streptomycetes are prolific producers of natural products, accounting for the majority of clinically used antibiotics and other bioactive compounds. Their remarkable biosynthetic diversity and ecological versatility are reflected in the large number of biosynthetic gene clusters (BGCs) within their genomes. However, many of these BGCs remain inactive under standard laboratory conditions, highlighting the need for high-throughput cultivation approaches to prioritise conditions that reveal strain- and medium-dependent metabolite production. Here, we present a microdroplet array platform that enables high-throughput cultivation and screening of Streptomyces. The array consists of 10,450 stationary nanolitre droplets, fixed onto a glass slide, that can be monitored for growth, morphology, bioactivity, and metabolite production. Streptomyces strains exhibited robust and reproducible growth while broadly preserving key strain-specific morphological features. Antimicrobial activity was assessed through nano-injection of Bacillus subtilis, revealing strain- and medium-specific antimicrobial activity. Strong inhibition was observed for Streptomyces sp. MBT27 when grown in TSB and MMMP media, with additional bioactivity detected for several strains when grown in MMMP. Single-droplet MALDI-TOF MS analysis of MBT27 grown in minimal medium droplets detected actinomycin X2, linking droplet-based phenotypes to chemical output. Follow-up cultivation under identical conditions in bulk cultures confirmed both antimicrobial activity and metabolite production by bioassays and LC-MS. These results demonstrate that the microdroplet array provides a high-throughput platform to monitor condition-dependent growth, morphology, and secondary metabolism. The system represents a promising upstream prioritisation tool for comparative cultivation studies and functional bioactivity screening in Streptomyces.
Abstract Salinity stress is a major abiotic constraint limiting crop productivity through osmotic imbalance, ionic toxicity, oxidative damage, and structural disruption. Here, we report a novel nano-enabled microbial cell factory system based on iron nanoparticle (FeNP)-loaded plant growth-promoting endophytic bacteria (PGPEB) isolated from Zea mays roots and identified as Bacillus sp. (16S rRNA accession MZ700077.1; 99.41% similarity). This living nano-bio hybrid was designed to integrate microbial plant growth-promoting functions with nanoparticle-mediated micronutrient delivery for enhanced salinity stress tolerance. Transmission electron microscopy confirmed successful formation of the microbial cell factory, revealing rod-shaped bacterial cells with intact flagella and uniform spherical FeNPs (~ 29.9 nm) strongly associated with the bacterial surface, indicating stable nano-bio interfacing without compromising cellular integrity. The engineered FeNP-PGPEB system significantly improved maize seed germination, root and shoot growth, and overall seedling vigor under moderate to severe salinity (200 mM NaCl), outperforming either PGPEB or FeNPs alone and demonstrating clear synergistic effects. Physiological and biochemical analyses revealed that the nano-bio formulation enhanced antioxidant defense systems (SOD, CAT, POD), increased proline accumulation, and optimized phytohormonal balance by elevating IAA and moderating ABA levels. In addition, lipid peroxidation (MDA) was reduced, while relative water content was maintained, indicating improved redox homeostasis and osmotic regulation. Ultrastructural and anatomical observations further confirmed reduced plasmolysis, improved membrane integrity, and enhanced vascular and root tissue organization under salt stress. Multivariate analyses (PCA and clustering) clearly distinguished treatments, with FeNP–PGPEB strongly associated with stress resilience traits and improved physiological stability. Overall, this study establishes a novel microbial cell factory platform, where endophytic Bacillus acts as a living nanocarrier for iron delivery, integrating microbial biotechnology and nanotechnology into a unified strategy for enhancing crop tolerance to salinity stress and advancing sustainable agricultural systems.
CDC15 is an important gene involved in mitotic exit and cytokinesis in S. cerevisiae. However, its additional functions remain unclear. Comparative genomics analysis between the fast-growing strain MC15 and the high-ethanol-producing strain MF01 revealed CDC15 as a significant sequence-divergent gene. Given this divergence, we hypothesized that replacing CDC15 in MF01 with the MC15 allele could enhance growth rate—a key trait for industrial production strains, as higher cell density reduces microbial contamination, shortens fermentation cycles, and improves overall efficiency. The CDC15 mutation significantly improved cell viability on solid medium at 50–54 °C. In liquid culture, the MT entered the stationary phase 8 h earlier than WT. Furthermore, the maximum cell densities achieved by MT at 30 °C, 37 °C and 41 °C increased by 37, 57, 155
The introduction of heterologous pathways into microbial hosts often imposes a metabolic burden on the cell, arising from three major physiological constraint layers: competition for gene expression resources, limited precursor availability and flux distribution, and insufficient energy and redox supply. Although Pseudomonas putida KT2440 is considered a robust and metabolically versatile production host, it remains unclear which of these constraint layers primarily limits heterologous terpenoid production in this organism. Here, lycopene biosynthesis was used as a model system to systematically dissect these three potential sources of metabolic burden. A capacity-monitoring system revealed no clear reduction in transcriptional or translational capacity across the tested strains and cultivation conditions, indicating that general gene expression capacity was not the primary limiting factor. Instead, lycopene production depended strongly on promoter architecture and plasmid backbone, showing that regulatory design shaped pathway performance. Enhancing precursor supply by introducing a heterologous mevalonate (MVA) pathway substantially increased product titres, identifying precursor availability from the native MEP pathway as the dominant bottleneck. This conclusion was independently supported by exogenous mevalonate supplementation, which further increased lycopene accumulation but also revealed saturation at higher concentrations, suggesting that downstream pathway balance or enzyme capacity became limiting once precursor supply was relieved. Under controlled bioreactor conditions, lycopene titres increased from approximately 1 mg/L to nearly 25 mg/L, indicating that process conditions further modulate production performance, suggesting an additional contribution of process-dependent energy and redox constraints. Metabolic burden during heterologous lycopene production in P. putida is governed primarily by precursor availability rather than by limitations in general gene expression capacity. Regulatory properties of the vector system strongly influence pathway performance, while controlled cultivation conditions can further improve production by alleviating additional process-dependent constraints. Together, these findings provide a systematic framework for distinguishing constraint layers and guiding the optimisation of heterologous terpenoid production systems.
Yeast surface display is a widely used platform for antibody affinity maturation; however, constraints in the yeast folding and disulfide bond formation machinery can limit correct antibody expression and bias selection outcomes, favoring variants that satisfy display constraints. This limitation might be of particular relevance when selections are based on biophysical features beyond affinity, such as aggregation, polyreactivity, or thermal stability. To overcome these constraints, we engineered the yeast display system by overexpressing key folding chaperones, yeast BiP and human protein disulfide isomerase (PDI), either through co-expression from the antibody display plasmid or via genomic editing, individually and in combination. As a proof of concept, surface display of adalimumab was significantly increased upon chaperone co-expression, with the highest improvement observed in strains with genomic integration of PDI, yielding a 2.5-fold increase in display levels. These findings were consistently reproduced across four additional antibodies using three edited strains expressing BiP, PDI, or both. To assess folding quality directly at the cell surface, we implemented two novel complementary staining strategies: with maleimide-Pacific Blue to detect unpaired thiols as a result of incomplete disulfide bond formation, and with Bis-ANS to quantify exposed hydrophobic regions. Both assays revealed a substancial reduction in free thiols and surface hydrophobicity (as only the proper hydrophobic residues are exposed) in the edited strains, consistent improved disulfide bond formation and overall folding quality relative to the parental strain. Accordingly, the PDI-edited yeast strain showed the best overall performance, improving both display quantity and quality across a panel of ten therapeutic antibodies. Enhanced display translated into improved antigen binding without altering the polyreactivity profiles of several candidates, therefore retaining native biophysical characteristics. The enhancement of the yeast folding machinery, particularly through genomic integration of PDI, substantially improves both the quantity and quality of antibody surface display. This optimized yeast display platform enables more faithful translation of antibody biophysical features, supporting its application in antibody workflows, including selections based on biophysical properties.
Nanobodies (NB) are compact single-domain antibody fragments with substantial diagnostic and therapeutic potential, but scalable microbial production under controlled bioreactor conditions remains insufficiently characterized. In this study a high-cell-density cultivation (HCDC) fed-batch process for an anti-human CD45 NB in Escherichia coli was established and evaluated how process parameters, host background, and subcellular targeting (cytoplasmic vs. periplasmic) affect yield and product quality including correct disulfide bond formation. Using a two-stage design-of-experiments (DoE) strategy in 2 L stirred-tank bioreactors, OD600nm at induction and post-induction temperature were identified as the main process variables controlling the post-purification nanobody concentration, whereas inducer concentration had no relevant effect within the tested range. Late induction at OD600nm 125–150 combined with production temperatures around 21–22.5 °C defined a robust operating window and yielded more than 2.5 g L−1 purified nanobody after Strep-tag affinity chromatography. Benchmarking of five host strain–plasmid combinations under standardized HCDC conditions further increased NB yield indicating that production performance was directly governed by the combined host background and expression architecture. While E. coli NEBExpress® with periplasmic SPPelB targeting reached around 2.4 g L−1 anti-hCD45 NB and the highest volumetric productivity (0.122 g L−1 h−1), E. coli SHuffle® T7 Express produced 2.2 g L−1 anti-hCD45 NB intracellularly. Nanobody-quality analysis revealed a pronounced difference between production routes. NB preparations obtained from periplasmic targeting constructs formed thermally uniform species with single nano differential scanning fluorimetry (DSF) unfolding transitions at 73.27–74.05 °C, whereas cytoplasmic derived preparations showed biphasic unfolding profiles with lower first transitions at 51.62–55.31 °C and second transitions at 68.30–71.53 °C indicating a higher thermal stability and also folding uniformity for secreted anti-hCD45 NB. Finally, flow cytometry confirmed functional binding of all tested anti-hCD45 NB to CD45-positive Jurkat cells. High-cell-density fed-batch cultivations of E. coli can reach > 2.5 g L−1 production of functional anti-CD45 nanobodies when induction timing, production temperature, host strain genetics, and subcellular location of the final product are aligned. The highest yield, most uniform, and thermally stable anti-hCD45 NB preparation was obtained with signal-peptide-mediated periplasmic targeting.
Thermostable and alkaline lipases are of significant interest for industrial applications, particularly in detergents and food processing. This study aimed to isolate, clone, and express lipase-encoding genes from a potent bacterial source to produce a thermo-tolerant alkaline lipase with enhanced catalytic efficiency and practical applicability. Among several bacterial isolates, the most potent lipase producer was identified as Lysinibacillus fusiformis, and its 16 S rRNA sequence was deposited in GenBank (PP757498). Three lipase-encoding genes (est, est2, and lipA) were successfully isolated, cloned, and heterologously expressed in Escherichia coli BL21 (DE3). Their sequences were submitted to GenBank under accession numbers PX136937.1, PX136938.1, and PX136936.1, respectively. The recombinant lipase encoded by lipA (rLipase) exhibited the highest activity (150 U/mL) compared with the native enzyme (56.2 U/mL). Molecular docking analysis demonstrated strong binding affinity of rLipase toward major fatty acid derivatives in olive oil, with the highest affinity for linoleic acid (− 8.0 kcal/mol), followed by oleic acid (− 7.8 kcal/mol) and palmitic acid (− 7.3 kcal/mol). These interactions were stabilized by hydrophobic interactions and hydrogen bonding, with key contributions from critical amino acid residues, particularly VAL250. The partially purified recombinant lipase (rLipase) exhibited a maximum activity of 320 U/mL at 80 °C and pH 9, demonstrating remarkable thermostability and alkaline tolerance. Functional evaluation showed that rLipase improved the detergent efficiency for oil stain-removal from cotton fabrics. In addition, supplementation with 0.4
Iron and other metal deficiencies in soil are significant challenges in agriculture, as almost 30
Abstract Background The utilization of endophytes as specialized microbial cell factories offers a sustainable and high-efficiency platform for the biosynthesis of functionalized nanomaterials. This study investigates the potential of a novel endophytic yeast, Clavispora lusitaniae , to produce cerium oxide nanoparticles (CeO₂NPs) and evaluates their efficacy against extensively drug-resistant (XDR) P. aeruginosa . Results An endophytic yeast, C. lusitaniae , was isolated for the first time from the medicinal plant Artemisia judaica and employed for the biosynthesis of CeO₂NPs, followed by surface capping with ethylene glycol (EG) to modify the nanoparticle surface properties. The synthesized nanoparticles were characterized using UV-Vis, FTIR, XRD, TEM, and DLS. These analyses confirmed the formation of spherical EG-CeO₂NPs with an average size of 8–20 nm. EG-CeO₂NPs exhibited strong antibacterial activity against XDR P. aeruginosa strains, with a minimum inhibitory concentration ranging from 0.6 to 1.25 mg/mL. Furthermore, these nanoparticles demonstrated potent anti-biofilm efficacy, achieving reductions of up to 89%. Mechanistic investigations demonstrated that EG-CeO₂NPs disrupt bacterial cell membranes, leading to intracellular protein leakage and elevated lipid peroxidation (indicated by increased malondialdehyde levels). Furthermore, the expression levels of quorum sensing (e.g., lasR and rhlR) and virulence-associated genes (e.g., toxA and exoS) were markedly downregulated by up to 87% compared to untreated controls. Conclusions This study establishes C. lusitaniae as a robust microbial cell factory for synthesizing functionalized CeO₂NPs with potent activity against XDR P. aeruginosa . Our results demonstrate the potential of microbial bioprocessing in engineering prospective nanotechnological platforms to combat antimicrobial resistance. However, further studies evaluating colloidal behavior under physiologically relevant conditions, mammalian-cell cytotoxicity, and in vivo efficacy are required before biomedical translation.
Heat stress is prevalent in industrial biomanufacturing processes including Baijiu brewing and bioethanol production. It disrupts the membrane structure and lipid homeostasis of Saccharomyces cerevisiae, interferes with genome-wide gene transcription and retards cell growth, thereby greatly lowering fermentation efficiency and product yields. There is an urgent demand for food-safe additives to alleviate heat-induced damage to industrial yeast. Although vitamin D₂ (VD₂) shares a similar sterol structure with endogenous yeast ergosterol, its regulatory effects on yeast under heat stress remain poorly understood. Here, we adopted an integrated strategy combining phenotypic characterization, lipidomics, transcriptomics and molecular docking to explore the protective effects and regulatory pathways of exogenous VD₂ against heat stress in S. cerevisiae. Phenotypic assays showed that VD₂ supplementation effectively alleviated heat-induced growth retardation, elevated intracellular glutathione peroxidase activity and reduced membrane injury. Lipidomic analysis revealed that VD₂ remodelled cellular lipid profiles by increasing triglyceride levels, decreasing phosphatidylcholine content and promoting sphingolipid synthesis. Transcriptomic results further demonstrated significant upregulation of key genes involved in the S-adenosylmethionine (SAM) metabolic axis. Integrated multi-omics data and molecular docking prediction support a tentative mechanistic hypothesis: VD₂ may interact with Opi3p and potentially interfere with its catalytic function, which could further drive downstream metabolic reprogramming. In conclusion, this study reveals that VD₂ alleviates heat-triggered growth suppression by remodelling the SAM-dependent lipid metabolic network. As a food-grade additive, VD₂ possesses promising potential to alleviate heat-induced growth retardation of yeast starters in industrial fermentation. Our findings provide a theoretical basis for developing natural additives to mitigate yeast heat damage and optimizing high-temperature fermentation bioprocesses.
Microbial robustness (i.e., to keep the same performance in the face of several perturbations) is a desirable trait for industrial yeasts, particularly in second-generation bioethanol production, where inhibitory compounds in lignocellulosic hydrolysates impair microbial performance. In this study, we investigated the role of the stress-responsive kinase Rim15 in stress adaptation and robustness by subjecting Saccharomyces cerevisiae wild-type (WT) and rim15Δ strain to adaptive laboratory evolution (ALE) in synthetic spruce hydrolysate (SSH). This hydrolysate contains high concentrations of inhibitory compounds, such as acetic acid and 5-hydroxymethylfurfural. Crucially, ALE consisted of two steps, including a medium swap regime that enabled adaptation to 100
Polyketides represent a broad class of structurally diverse natural products with potent antibacterial and anticancer activities. Type II polyketide synthases (T2PKS) are multi-subunit complexes responsible for condensing acylated intermediates, including acetyl-CoA and malonyl-CoA, into complex fused-ring aromatic natural products. While type II polyketides have traditionally been associated with gram-positive actinomycetes, more recent discoveries have identified PKS systems in gram-negative bacteria. These findings have provided new genetic tools and opened new pathways for producing polyketides in the versatile chassis organism Escherichia coli. In this study, we altered the genetic circuit of the Photorhabdus luminescens PKS for octaketide production in E. coli. Promoter engineering and ribosome-binding-site optimization of the antDEFBG minimal PKS (mPKS) in M9 minimal media resulted in SEK4 and SEK4b production titers of 36 mg/L and 78 mg/L in strain E. coli sAJM.1506/pANT7, respectively. Further refinement of downstream ketoreductase, aromatase, and cyclase expression enabled the biosynthesis of native polyketide AQ-256 and naphthopyrone (S)-DNPA reaching a highest measured titer of 141.3 ± 9.2 mg/L after 24 h of shake-flask cultivation. Overexpression of the P. luminescens malonyl-CoA acyl carrier protein transacylase and acetyl-CoA carboxylase complex further improved production by 50
This review examines the current state of single-cell protein (SCP) research based on waste-derived substrates in Nigeria, with emphasis on substrate choice, microbial systems, bioprocess strategies, performance reporting, and prospects for commercial translation. Evidence from Nigeria-focused studies shows that a broad range of locally available waste streams, particularly those containing cassava peels, yam peels, cereal byproducts, fruit wastes, and sawdust, can support microbial growth and protein content improvement when yeasts, filamentous fungi, bacteria, and, to a lesser extent, mixed cultures are used. Most studies clearly demonstrate biological feasibility, and several also report reductions in the levels of fibre, cyanide, or other antinutritional factors. A relatively small number of studies extend to short-term feeding trials, providing early support for feed-oriented application. Despite this promise, the Nigerian literature remains dominated by laboratory-scale studies based largely on simple solid-state or batch submerged fermentation systems. Important translational elements, including strain robustness, process standardisation, downstream recovery, local techno-economic assessment, and regulatory preparation, remain poorly developed. Inconsistently reported protein-related outcomes have been reported, with many studies relying on crude or soluble protein measurements without a deeper assessment of amino acid quality, digestibility, or product safety. As a result, a clear gap persists between demonstrated laboratory feasibility and market-ready SCP production.
Mycolicibacterium strains are among the most effective biofactories for converting phytosterols into active pharmaceutical intermediates. Despite the main route resembling fatty acid β-oxidation, the specific enzymes and their precise roles are poorly defined. In this study, we performed a comprehensive gene knock-out analysis in the industrial 4-AD-producing strain, Mycolicibacterium neoaurum HGMS9 strain, a variant of M. neoaurum B-3805. Our results demonstrate that the three β-oxidation cycles responsible for phytosterol side-chain degradation share a suite of core enzymes. Specifically, seven enzymes, including the acyl-CoA dehydrogenase ChsE1/E2, ChsE4/E5, the hydratase, ChsH1/H2 and the aldolase Ltp2, function in both the second and third β-oxidation cycles to generate the final products, 4-AD. Importantly, we identified five potential secondary routes that divert metabolism to produce eight off-route C22, C23 and C24 intermediates. These compounds represent valuable precursors for the synthesizing advanced steroids like corticosteroids and bile acids. This study not only clarifies the enzymatic steps of phytosterol side-chain degradation but also establishes a metabolic blueprint for engineering high-yielding Mycolicibacterium strains.
The Gram-positive bacterium Bacillus subtilis is a rapidly growing and easily manipulated microbe with a long history of exploitation for the commercial production of industrial enzymes, high value biochemicals, antibiotics and other secondary metabolites. More recently, it has been developed as a food additive and plant probiotic. It grows on inexpensive substrates and remains productive during extended batch-fed growth conditions. Extensive knowledge of its genetics, biochemistry and gene regulation has facilitated the use of metabolic engineering strategies to optimise substrate utilisation and product yield. As a free-living environmental organism, B. subtilis differentiates into subpopulations with distinct biological functions (e.g. sporulation, biofilm formation, production of antimicrobials, etc.). However, during fermentation, cellular differentiation processes can pose a challenge for its optimal biotechnological utilisation, particularly when emerging subpopulations do not contribute to product biosynthesis. Here, we present robust assays that facilitate the analysis of two previously difficult-to-study population properties of B. subtilis: (i) the energisation levels of individual cells within post-exponential but actively growing cultures and (ii) the extent of cell lysis that can occur under such conditions. Our findings reveal an unappreciated level of heterogeneity in cell energisation within post-exponential cultures, and a surprisingly high degree of cell lysis in seemingly healthy, actively growing populations. These data provide insights and add to our understanding of the biological complexities and single-cell heterogeneities present in superficially simple bacterial clonal cultures. They establish robust and well-validated analytical tools with which to study the associated processes and provide a foundation for further optimising B. subtilis as an industrial production host. Considerable research efforts have focused on increasing the productivity of B. subtilis for industrial, medical and agricultural products. However, its ability to undergo physiological and morphological differentiation processes at high cell densities could ultimately limit its productivity. Our research reveals how the resulting heterogeneity impacts the population-level energy status of individual cells in the culture and the surprisingly high extent of population-level cell lysis. The ability to monitor these processes provides tools for evaluating the impact of genetic and metabolic engineering strategies to improve productivity.
Copolyesters of (R)-3-hydroxybutyrate and medium-chain-length (R)-3-hydroxyalkanoate) [P(3HB-co-mcl-3HA)] are a highly flexible kind of polyhydroxyalkanoates (PHAs), although the efficient biosynthesis still remains a challenge. Biosynthesis of P(3HB-co-mcl-3HA) copolyesters was attempted using a class II PHA synthase in Cupriavidus necator H16, a well-studied PHA producer. Expression of the mutant of PHA synthase 1 from Pseudomonas sp. 61-3 (PhaC1EDQK), exhibiting broad substrate specificity to 3HA-CoAs of C4–C12, resulted in accumulation of only a small amount of PHA from soybean oil. This was caused by disappearance of PhaC1EDQK specifically in the soybean oil-grown cells, despite active transcription of the corresponding gene. Co-expression of pseudomonad phasins PhaIPs61-3 and PhaFPs61-3 tended to increase PHA production. During this investigation, we identified a spontaneously generated chimeric mutant of the two phasins (designated PhaIFchi), and found that PhaIFchi greatly increased P(3HB-co-mcl-3HA) accumulation on soybean oil. Further analysis revealed that both PhaC1EDQK and native phasin PhaP1 lacked binding ability to P(3HB-co-mcl-3HA) granules, which was likely associated with the low level of PHA production. In contrast, PhaIFchi, comprised of a fusion of PHA-binding domains and predicted to form a tetrameric structure, was able to not only bind to the granules but also assist in localization of PhaC1EDQK and PhaP1 on the granules, thereby promoting copolyester production. This study provides new insights into a novel function of granule-associated proteins in PHA biosynthesis, and demonstrates that engineering of phasins is a promising strategy for establishing efficient biomanufacturing of practical bioplastics.
Lactic acid bacteria and their metabolites have shown promising biological activities against different types of cancer, esspecially, colon cancer. In our previouse study, we extracted and optimized the production of a mixture of three oligosaccharides from Lactobacillus acidophilus DSMZ 20079 at the laboratory scale using statistical experimental designs and the active oligosaccharide HPLC fraction was identified as LA-EPS-20079. Also, the anticancer activity of the extracted LA-EPS-20079 against colon cancer CaCo-2 cell line was confirmed via the downregulation of the gene expression of Bcl2 and Survivin genes (6). In the present study, we produced a mixture of bioactive oligosaccharides from L. acidophilus DSMZ 20079 in 10 L bioreactor that maintained the anti-colon cancer against CaCo-2 cell line effects, which is a major transition towards the semi industrial level. To that end, a batch fermentation was carried out in a 10 L bioreactor under uncontrolled pH condition, starting at pH 6.0. During the fermentation process, the bacterial cell mass was increased exponentially with a constant specific growth rate (µ) of 0.07 h−1, reaching a maximum biomass of 6.93 g L−1 at 44 h. Also, the concentration of the produced total exooligosaccharides gradually increased and reached a maximum concentration of 1461.43 µg mL−1 at 40 h post-incubation. Furthermore, the glucose consumption rate was 1.21 g L−1 h−1, while the LAB-oligosaccharide production rate reached 96.22 µg mL−1 h−1. Moreover, oligosaccharides chemical composition was studied after scaling up the production process using 1D and 2D proton and carbon NMR, along with monomeric composition analysis. The results indicated that LAB-oligosaccharide is a mixture of oligosaccharides, not a single structure, and the mixture consists of three monomeric sugars; herein, we referred to the oligosaccharide mixture as LAB-oligosaccharide. Additionally, the anticancer activity of LAB- oligosaccharide was tested in samples collected from the bioreactor at 0, 12, 14, 16, 18, 20, 22, 24, 36, 40 and 44 h time points against the CaCo-2 cancer cell line. The results confirmed the in-vitro anticancer activity with maximum inhibition percentages of 72.01
Abstract The filamentous actinomycete Actinomadura namibiensis has attracted increasing interest as the only known natural producer of the carbocyclic lantibiotic labyrinthopeptin A1, a secondary metabolite with a broad antiviral spectrum of activity. Developing a model of microbial growth and product formation at the pellet level requires a deeper mechanistic understanding of the interactions between cultivation conditions, substrate availability, and oxygen limitation. These factors influence the cultivation process and product formation. As part of this work, a predictive model in the form of diffusion–reaction equations is developed to quantitatively describe nutrient uptake, oxygen diffusion limitation, and pellet growth. This framework couples the consumption kinetics of carbon sources, such as glucose and glycerol, to pellet growth, maintenance metabolism, and the formation of the secondary metabolite labyrinthopeptin A1. The model is calibrated based on the results of multiscale experiments, i.e., offline analysis of shake flask cultures and oxygen profiling of pellets at different time points under various conditions. This forms the basis for understanding nutrient uptake, metabolic heterogeneity, and mass transfer limitations within the pellets. Thus, the model provides a quantitative basis for predicting productivity. The validated multiscale model reproduces the experimental results well and can be used to predict pellet growth, substrate uptake, and product formation.
Microagglutination is a sensitive, rapid, and cost-effective serological assay widely used for disease diagnosis and surveillance in animals and humans. However, it traditionally relies on culturing live pathogenic bacteria as antigens, posing biosafety risks, requiring specialized infrastructure, and limiting its application primarily to bacterial pathogens. Bacterial surface display technology offers a promising alternative by enabling the presentation of heterologous proteins or peptides on the bacterial surface, avoiding the need to culture the target pathogen, simplifying antigen preparation, and enabling potential diagnostic applications for non-bacterial pathogens. Despite these advantages, integration of this technology with slide-based microagglutination assays has not yet been evaluated. In this proof-of-concept, we evaluated a surface display platform in attenuated Salmonella Enteritidis for antibody detection by slide microagglutination, using the nucleocapsid (N) protein of Infectious Bronchitis Virus (IBV) as a model antigen. The IBV N protein was fused to the N-terminal anchor domain of the ice nucleation protein (INP) and cloned into pET-28a. The resulting INP_N fusion protein was expressed in attenuated Salmonella Enteritidis, and its surface exposure was evaluated by immunofluorescence and flow cytometry on non-permeabilized cells, with flow cytometry indicating a surface display efficiency of 96.5
Citric acid, a high-volume bioproduct, is manufactured by submerged fermentation using the filamentous fungus Aspergillus niger. Citric acid overflow requires Mn(II) ion concentrations not exceeding 5 µg L− 1. However, manganese easily leaches from media ingredients or stainless-steel bioreactors, necessitating costly, laborious removal. Aspergillus niger citrate fermentations were evaluated across varied manganese profiles, comparing a novel double transporter mutant against parental and single-mutant strains. This strain overexpresses cexA – the primary plasma membrane exporter whose activity overcomes a major production bottleneck – under the control of the strong glaA promoter in a background deficient in dmtA, which encodes the primary high-affinity NRAMP-family manganese transporter. Under high-manganese conditions (100–165 µg L− 1) simulating industrial substrate impurities and bioreactor leaching, the double transporter mutant outperformed the other strains. Despite abundant manganese that triggers high biomass (50 g L− 1) and low citrate yields in the parental strain NRRL2270, this cell factory maintained high-yield overflow metabolism. Driven by an accelerated maximal glucose consumption rate of 1.61 g L− 1 h− 1 and reduced biomass accumulation (9.1 g L− 1), it accumulated citric acid to levels comparable to those achieved under manganese deficiency. The double transporter mutant compressed the fermentation period by 17
