Applied Microbiology and Biotechnology

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.

Garvicin KS (GarKS) is a three-peptide, leaderless, broad-spectrum bacteriocin that is active against a wide range of Gram-positive bacteria, including several foodborne pathogens and antibiotic-resistant strains. This bacteriocin is considered a candidate for application in food preservation and medical treatments; however, key knowledge about the producer strain, regulation of production, and the mechanism of action is still lacking for leaderless, multipeptide bacteriocins, including GarKS. A hybrid sequencing strategy was used to obtain a high-quality closed genome assembly of the native GarKS producer Lactococcus garvieae KS1546, which showed that the bacteriocin was encoded on a 50 kb plasmid (pKS50). Comparative analysis with updated sequence databases indicates that the producer strain should be reclassified as Lactococcus petauri. The roles of the biosynthetic genes, putatively encoding a transcriptional regulator (gakR) and immunity protein (gakI), were examined using heterologous expression. Removal of gakR resulted in a 6-fold decrease in GarKS production, and expression of gakI caused a 250-fold decrease in susceptibility to GarKS, with varying degrees of cross-immunity to other multipeptide bacteriocins. Isolation and whole-genome sequencing of spontaneous GarKS mutants in L. lactis showed that resistance levels are low, but that ythA, a PspC-domain-containing protein involved in a phage stress response pathway is involved in the GarKS susceptible phenotype. To decipher conserved features involved in production of these bacteriocins, we finally used genome mining to identify 11 candidates for new multipeptide bacteriocins; four of them were obtained synthetically and confirmed to be bioactive peptides inhibiting important pathogens including Listeria monocytogenes and enterococci.

Lager is the most produced beer style world-wide and makes use of the bottom-fermenting hybrid yeast Saccharomyces pastorianus (S. cerevisiae x S. eubayanus). Previous research showed that flocculation in S. pastorianus, in contrast to the top-fermenting ale yeast S. cerevisiae, is triggered by nitrogen starvation. However, the cellular events leading to flocculation in S. pastorianus are not well characterized. Therefore, we conducted a proteomic screen of S. pastorianus TUM 34/70 and identified the protein kinase Ste20p and protein phosphatase regulatory subunit Ypi1p as higher abundant during flocculation. Overexpression of these genes caused a consistent and strong increase in flocculation rate over the complete duration of beer fermentation. Characterization of Ste20p and Ypi1p via a phospho-proteomics screen showed their targeting of proteins whose S. cerevisiae orthologues are involved in pseudohyphal growth. However, in contrast to this, the overexpression of STE20 and YPI1 led instead to the establishment of a flocculation morphology, giving first-time evidence that S. pastorianus repurposes the pseudohyphal signaling network for this phenotype.

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.

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.

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.

Biopreservatives including nisin and its derivatives are becoming more desirable in the food processing industry because of the growing demand for naturally preserved and minimally processed foods free from artificial preservatives. However, ensuring microbiological safety while meeting these consumer preferences remains a major challenge. This has necessitated the continuous investigation of potential new antimicrobial agents produced by naturally occurring microorganisms. Hence, this study explored the synthesis, characterisation, and optimisation of a bacteriocinogenic lactic acid bacterium and its antimicrobial product, possibly novel bacteriocin (Nisin 2A) from Lactococcus lactis isolated from commercial brined cheese. The isolation was achieved by screening for wild-type bacteriocin-producing lactic acid bacteria from dairy products using MRS media. Screening was performed using antagonism assays, yielding five producer organisms. Of these, the isolate whose metabolites exhibited the most potent antimicrobial activity was identified as Lactococcus lactis, which synthesised an active antimicrobial peptide designated as Nisin 2A, with a molecular mass of approximately 3.3 kDa as determined by UHPLC-MS and SDS-PAGE. Production of Nisin 2A was scaled up through fed-batch fermentation of Lactococcus lactis in modified MRS broth following process optimisation using a Plackett-Burman experimental design and purified by ammonium sulphate precipitation and solid-phase extraction (SPE). Furthermore, the antimicrobial potential of the bacteriocin was evaluated by the agar well diffusion assay and quantified using the tube dilution method. The purified peptide demonstrated broad-spectrum antimicrobial activity, particularly against the test Gram-positive bacteria Bacillus cereus and retained its bioactivity across a wide pH range (3-9) and high thermal conditions (up to 100 {degrees}C). Furthermore, it had high sensitivity to proteolytic enzymes (Proteinase K and Trypsin). Notably, the peptide was thermostable and retained up to 90% of its initial activity after thermal treatment and maintained consistent inhibitory performance after extended storage. These findings highlight the potential application of Nisin 2A as a natural biopreservative in food systems.

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.

In the past decades, several tools to genetically modify the human pathogen Listeria monocytogenes were developed. Here, we constructed a new integrative plasmid system for L. monocytogenes named pTRIP, for treB insertion plasmid. pTRIP is a vector which stably integrates into the treB locus of the wild type EGD-e. This locus encodes the sole trehalose-specific EIIB and EIIC component of a phosphotransferase system. Successful integration leads to the disruption of treB and thus, to an inability of the resulting L. monocytogenes strains to grow on trehalose as sole carbon source. Due to integration through double homologous recombination, it is the first integrative system which does not require antibiotic selection pressure. To assess functionality of the pTRIP system, prfA and its native promoter region were integrated into the treB locus of a {Delta}prfA strain. Complementation was confirmed in 78% of the isolated clones, indicating successful integration of prfA into the treB locus. We further constructed derivatives of pTRIP harboring the constitutive Pp60 (pTRIP1) and the inducible Prha (pTRIP2) promoter to further expand application possibilities. Microscopic analyses confirmed the functionality of both promoter constructs and showed dose-dependent induction for Prha. pTRIP is an efficient tool for stable gene expression as well as functional studies and expands genetic modification possibilities for L. monocytogenes.

Trace elements, such as iron, nickel, and cobalt are known to regulate methanogenic activity in anaerobic digestors used for waste valorisation, but the potential role of rare earth elements remains poorly understood. This study investigated the effects of lanthanum (La) supplementation on biogas production, methane generation, volatile fatty acid (VFA) formation, and carbohydrate utilisation in anaerobic digestion (AD). Biomethane potential (BMP) assays conducted under mesophilic conditions (37C) using methanogenic sludge granules, and glucose as substrate, were supplemented with 0.1, 1, 10, and 100 mg/L lanthanum chloride (LaCl3). Biogas production and composition was monitored over a 96-h incubation, while sacrificial, batch bioreactors were used to evaluate temporal VFA and carbohydrate profiles. La supplementation significantly enhanced biogas and methane production in a concentration-dependent manner. The highest cumulative biogas yield (478.9 mL, corresponding to 179.5 mL biogas/g COD) and methane production (285.7 mL, corresponding to 107.1 mL CH4/g COD) were observed with 100 mg/L LaCl3, corresponding to increases of 88.7% and 186%, respectively, compared with La-free controls. CO2 production also increased with La concentration, whereas hydrogen production remained comparatively low. Acetic and butyric acids represented the dominant fermentation products (80-88% of total VFAs), but profiles of accumulated VFA in the bioreactors diversified with La addition, including showing caproate production, indicating changed biodegradation dynamics in the methanogenic microbiome. These findings demonstrate that lanthanum can stimulate anaerobic digestion performance and methane generation, highlighting the potential as a novel trace element additive to enhance biogas production. Research is now required to elucidate the underlying microbial and biochemical mechanisms, and establish optimal dosing strategies for large-scale applications.

This study investigates electro-fermentation candida Tropicalis SY005 to address fermentation kinetics limitation during grape must fermentation. In comparison with non-stimulated control sample, EF substantially enhanced sugar depletion, TSS drop by day 3 and generated a strongly reduced state (ORP -100 to -143mV). The oxidation-reduction shift enhanced cellular NAD+ regeneration, reducing total fermentation duration from 264 h to 72 h. GC-MS analysis showed pronounced major characteristic volatile compound confirming substantial metabolic pathway shifts in flavor of glycolytic flux. Moreover moderate electric field promoted cellular membrane electropermeabilization substantially promoting bioactive extraction.

Given the current global environmental challenges, waste biomass is an attractive renewable resource for circular economies. Gasification of biomass yields syngas (CO, CO2, and H2) that is a suitable feedstock for gas fermentation in biomanufacturing of fuels and chemicals using acetogen microbes. While it is generally known that syngas composition influences both acetogen growth and process performance, we are lacking a consistent dataset quantifying these effects under controlled fermentation conditions. Here, we mapped the metabolic response of the model-acetogen Clostridium autoethanogenum to seven synthetic syngas mixtures during exponential batch growth in bioreactor fermentations. Notably, distinct gas compositions resulted in different fermentation profiles, affecting both growth and metabolite production. Maximum specific growth rates ranged within 0.05 0.13 h-1, with slower growth for low-CO mixtures. While acetate and ethanol production yields varied between 20-133 and 76-353 mmol per gram dry cell weight, respectively, minor production of 2,3-butanediol was detected. All syngas mixtures supported co-utilization of CO and H2, though gas uptake stoichiometry only moderately correlated with syngas content. Importantly, gas uptake stoichiometry strongly influenced carbon partitioning, with higher relative H2 uptake reducing CO2 loss or even realizing CO2 fixation together with increasing carbon flow towards metabolites. Interestingly, higher syngas H2 content favored ethanol and 2,3-butanediol production, while higher H2:CO uptake ratios increased total flux through the Wood-Ljungdahl pathway rather than selectively favoring reduced by-products. Our results are valuable for a better understanding of syngas composition effects on the acetogen biocatalyst and for process engineering towards optimizing gas fermentation performance. HighlightsO_LISyngas composition affects acetogen growth, gas uptake, and carbon distribution C_LIO_LIHigher H2:CO uptake ratios increase carbon flow through the Wood-Ljungdahl pathway C_LIO_LIHigher relative H2 uptake reduces CO2 loss and increases metabolite production C_LI

Acetate is a promising carbon source for microbial biotechnology as it can be produced sustainably from lignocellulosic biomass or C1 gases. Since acetate is directly activated to acetyl-CoA, it is especially suitable for producing acetyl-CoA-derived products, showcased here with the production of 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs). P. putida KT2440 can natively metabolize acetate, but the weak acid has also inhibitory effects on microbial growth. We present an in-depth study on the physiology of P. putida KT2440 using acetate as carbon and energy source and evaluate acetate as feedstock for the biosynthesis of HAAs. Initially, a rational engineering approach to overexpress acetyl-CoA synthetase for acetate activation resulted in an improved growth rate of 16% and reduced lag phase by six hours. To further increase the performance of P. putida KT2440 on acetate, adaptive laboratory evolution was performed. This resulted in an improvement in the growth rate from 0.4 h-1 to 0.6 h-1 and enabled growth on up to 12.5 g L-1 acetate with a shortened lag phase compared to the wild type. Whole-genome sequencing revealed mutations in proteins involved in gene expression regulation and signal transduction. This evolutionary engineering approach informed the deletions of gacS and crc, which resulted in a reduction in the lag phase from seven hours to one hour and an improvement of the growth rate by 25 %, matching the growth properties of the evolved clones. Using the evolved strains for the production of HAAs resulted in faster biomass and product formation with product titers reaching up to 94 % of that of the wild type. In conclusion, we identified mechanisms in the acetate metabolism of P. putida KT2440 and improved the growth performance of the strain by rational and evolutionary engineering, demonstrating the potential of the promising, but challenging 3rd generation feedstock acetate.

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.

Spices contain diverse plant secondary metabolites with antimicrobial and antioxidant activities that can influence plant-microbe interactions. Among these metabolites, curcumin and piperine are major bioactive constituents of turmeric and black pepper, respectively. In this study, we demonstrate that curcumin and piperine enhance alcoholic fermentation in the budding yeast Saccharomyces cerevisiae. Curcumin significantly promoted alcoholic fermentation under high-glucose conditions (20-40% glucose), which impose substantial osmotic stress on yeast cells. Under these conditions, curcumin enhanced yeast growth, improved osmotic stress tolerance, and increased resistance to cell wall digestion, suggesting enhanced stress adaptation during fermentation. Furthermore, deletion of HOG1, which encodes the central MAP kinase of the High-Osmolarity Glycerol (HOG) pathway responsible for the osmotic stress response, abolished the fermentation-enhancing effect of curcumin, indicating that Hog1 function is required for this effect. Consistent with this result, transcriptomic and RT-qPCR analyses revealed increased expression of stress adaptation-related genes, including known HOG pathway-responsive genes. In contrast, the expression of genes involved in amino acid catabolism was decreased at 12 h after the initiation of fermentation, while metabolomic analysis revealed increased intracellular levels of amino acids, including glutamate and asparagine, at 24 h. These coordinated transcriptional and metabolic changes are consistent with reduced amino acid catabolism and a shift toward a metabolic state favorable for sustained fermentation. Together, these findings suggest that curcumin enhances alcoholic fermentation under osmotic stress by promoting stress adaptation that supports yeast growth and sustained fermentation.

Polycyclic aromatic hydrocarbons (PAHs) are hazardous organic contaminants for which microbial bioaugmentation is a promising remediation strategy, but poor persistence of introduced microorganisms can limit efficacy. Encapsulation may improve persistence, yet the influence of capsule design, microbial species, and environmental conditions on performance remains poorly understood. We evaluated alginate encapsulation of the PAH-degrading bacteria Pseudomonas putida and Novosphingobium aromaticivorans across nutrient conditions and capsule formulations. Encapsulation effects varied by species and medium, influencing growth rate, maximum cell density, overall growth, and lag time; notably, encapsulation shortened lag time of N. aromaticivorans in sRB15 medium (36.9 h to 3.9-5.3 h). Enumeration methods also affected apparent cell recovery. After 8 weeks, encapsulation had no significant effect on P. putida but resulted in increased concentrations of N. aromaticivorans relative to planktonic cultures (1.22 x 10; vs. 2.05 x 10; CFU/mL). Capsule composition further influenced cell retention: increasing alginate approximately doubled capsule-associated cell concentrations, while chitosan coatings reduced cell concentrations within capsules without affecting external concentrations. These findings demonstrate that the benefits of encapsulation are species- and environment-dependent and that capsule formulation can be tuned to influence bacterial persistence and release, informing the design of encapsulated inoculants for bioaugmentation applications.

Aims Honey represents a distinct microbial habitat, imposing physicochemical stresses on its resident microbiota while also serving as a potential source of beneficial microorganisms. This study aimed to isolate lactic acid bacteria (LAB) from Korean wildflower honey and characterize their gastrointestinal (GI) and physiological stress tolerance, adhesion-related properties, functional characteristics, and safety to evaluate their potential as probiotic candidates. Methods and Results Two Streptococcus salivarius strains, DOW-5A and DOW-10, were isolated from Korean wildflower honey and identified by phenotypic and molecular analyses. Both strains survived in sequential exposure to simulated GI conditions and showed tolerance to physiological stresses. DOW-5A maintained higher viability during the simulated gastric phase, whereas DOW-10 showed an initial decrease in viable counts followed by an increase during the simulated intestinal phase. The two strains also exhibited various probiotic characteristics, including mucin adhesion and antioxidant activity. Atypical, crystalline colony morphologies associated with EPS production were observed when sucrose was supplied as a carbon source. Both strains formed biofilms, while DOW-5A showed a marked increase in biofilm formation upon sucrose supplementation. Neither strain showed gelatinase or hemolytic activity or significant cytotoxicity toward Caco-2 cells. Impact Statement In this study, we isolated two honey-derived S. salivarius strains with high probiotic potential. Our findings highlight wildflower honey as a potential reservoir of LAB with traits relevant to survival under GI and physiological stresses, as well as various probiotic traits.

Nisin is a promising antimicrobial peptide widely used in food preservation due to its efficacy against Gram-positive spoilage and pathogenic bacteria. Although Nisin is increasingly applied in the food sector, the biopeptide suffers from instability within food matrixes and can rapidly lose its antimicrobial potential following interaction with food biomolecules. Thus, it is necessary to investigate approaches that can be employed to extend the stability and activity of Nisin. Hence, the aim of this study was to develop and characterise a chitosan-alginate polyelectrolyte microencapsulation system capable of enhancing Nisin stability while retaining antimicrobial activity. The microencapsulation of Nisin was achieved by pre-gelation of alginate using calcium chloride and subsequent direct electrostatic interaction between cationic Nisin and chitosan with pre-gelled anionic alginate at pH 5.0. Following microcapsule formation, physicochemical and structural characterisation was performed using Zeta potential determination and measurement of the polydispersity index (PDI) via dynamic light scattering. SEM micrographs were used to confirm morphology, while Fourier-transform infrared (FTIR) spectroscopy and high-performance liquid chromatography (HPLC) were utilised to assess chemical integrity and functional group preservation of encapsulated Nisin. Following this, stable microcapsules with diameters ranging from 150-200 nm and smooth surface morphology were obtained. Microcapsule formation was strongly influenced by formulation parameters, particularly pH, calcium ion concentration, and chitosan content, with deviations from optimal acidic conditions (< pH 5.0) resulting in aggregation, increased polydispersity, and reduced encapsulation efficiency. The microcapsules were monodispersed (PDI {approx} 0.30) and electrostatically stable, exhibiting a Zeta potential of approximately +36 mV. These microcapsules remained stable over a prolonged storage period of 21 days under refrigerated conditions while retaining antimicrobial activity against Bacillus cereus. Encapsulation efficiency reached approximately 65%, confirming effective retention of Nisin within the polymer matrix. Overall, the findings demonstrate that chitosan-alginate ionic gelation is a non-denaturing and effective encapsulation strategy for extending the functional stability of Nisin. These microcapsules show strong potential as natural antimicrobial delivery systems for food and beverage applications, particularly in acidic food matrices, with implications for improved food safety and shelf-life extension.

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.

The growing concerns due to plastic pollution in India have intensified the search for sustainable materials. Mycelium-based composites (MBCs) have emerged as bio-based alternatives for packaging and thermal insulation applications. India, the worlds largest producer of bananas, generates significant quantities of banana biomass (~200 tons per hectare per year), much of which remains underutilized. The banana peduncle, the stalk that supports the fruit bunch, is one such underutilized biomass. In this study, banana peduncle fibers were used as the main substrate with Pleurotus ostreatus for the fabrication of MBCs. Banana peduncle fibers were mixed with wood shavings (10-50 wt%) to enhance the dimensional stability and structural integrity of the composites. The properties of developed MBCs such as density, shrinkage, moisture absorption, water absorption, morphology, compressive properties, and thermal conductivity were studied. The 90% banana peduncle fibers-10% wood shavings formulation showed the highest radial mycelial growth rate (7 mm/day). MBCs consisting of 100% banana peduncle fibers had volumetric shrinkage of 36%, while the incorporation of 30-50% wood shavings reduced shrinkage by approximately 17%. Among the formulations, MBCs containing 30% wood shavings had the highest compressive strength (4.82 MPa) and compressive modulus (1.78 MPa), whereas MBCs containing 50% wood shavings had the highest recovery (59.6%). In contrast, MBCs fabricated using 100% banana peduncle fibers had the lowest thermal conductivity (0.04 W/m. K). These results demonstrate that banana peduncle fibers are a promising lignocellulosic substrate for the development of MBCs for sustainable packaging and thermal insulation.