The overexpression of proteins in Escherichia coli often results in the formation of inclusion bodies, which are biologically inactive, especially for proteins with exposed hydrophobic surfaces. Solubilization of inclusion bodies (IBs) and subsequent refolding is essential for obtaining correctly folded and active protein. However, protein refolding involves multiple steps—namely isolation, solubilization, and refolding—which is a labor-intensive process. In this study, we developed a strategy for soluble production and protein refolding. A fusion tag was applied to Burkholderia ambifaria lipase YCJ01, enabling abundant soluble expression in E. coli. Despite this, the soluble protein exhibited only partial enzymatic activity, suggesting an unfolded state of soluble lipase YCJ01. Lipase activity increased significantly after incubation with cosolvents, reaching 1003 U/mL, 754 U/mL, and 501 U/mL in 25% (v/w) glycerol, 15% (v/w) DMSO, and 4M trimethylamine N-oxide (TMAO) solutions, respectively. Correctly folded and highly active lipase YCJ01 with a natural N-terminus was obtained. Moreover, the cosolvent-induced refolding mechanism was elucidated through molecular dynamics simulations. Glycerol and DMSO were found to aggregate around hydrophobic regions of lipase, directly stabilizing structure by displacing water molecules and weakening water–protein hydrogen (H) bonds within the hydration shell. Conversely, TMAO molecules indirectly influenced the lipase structure by strengthening water–water H bonds.Key points• Cosolvents enhance lipase activity, with glycerol showing the highest improvement.• MD simulations show glycerol and DMSO directly interact with hydrophobic regions.• Glycerol and DMSO stabilize lipase directly, while TMAO enhances stability indirectly.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-025-13660-6.

Honey bees gather pollen from flowering plants, using it as a vital protein source and, in turn, acquire pollen-associated microbes that interact with their existing gut microbiota. Despite their ecological importance, limited information exists regarding the gut microbiota of African savannah honey bees (Apis mellifera scutellata Lepeletier) and how diet and its associated microbial community influence this crucial internal ecosystem. This study aimed to investigate the differences in gut microbiota between wild honey bees collected during the flowering season and microbially depleted honey bees reared under semi-sterile conditions and fed various protein diets. To achieve this, freshly hatched worker bees were maintained in hoarding cages and assigned one of four protein diets: fresh sunflower pollen, casein, sterilised casein, or sterilised pollen. High-throughput DNA metabarcoding was then employed to compare the microbial composition of the honey bee gut across these groups. Our findings revealed that the gut of microbially depleted honey bees exhibited higher species diversity and richness. Conversely, the non-core gut microbial community predominated in wild bees and those fed the different protein diets. Specifically, Commensalibacter, Bartonella, and Bifidobacterium were the most dominant bacterial genera across all treatments. Interestingly, Gilliamella, a common core gut bacterium, was undetected, while Apibacter was exclusively found in wild honey bees. Furthermore, pollen-associated microbes such as Devosia and Pedobacter were identified solely in the gut of honey bees fed a pollen diet. Functional predictions of the gut microbial community also indicated the presence of key enzymes such as β-glucosidase, β-galactosidase, pyruvate dehydrogenase and phosphoglycerate mutase, which are crucial for enhancing nutrient absorption, digestion, and carbohydrate metabolism. These results underscore the intricate relationship between honey bees, microbes, and plants, offering valuable insights into how diet and its associated microbial communities could shape the gut microbiota of African honey bees. KEY POINTS: • The non-core gut microbiota dominates the African savannah honey bee •The type of diet influenced the microbial diversity and community abundance in the honey bee gut •Key enzymes involved in digestion, nutrition absorption, and carbohydrate metabolism were enhanced in the gut •Pollen-associated microbes found in the diet present potential avenues for probiotic development to improve honey bee health.

Streptomyces are renowned in pharmaceutical and medical fields for their ability to produce antibiotics and other bioactive secondary metabolites. In order to reduce industrial production costs, it is crucial to find suitable and cheaper raw materials as carbon and nitrogen sources for microbial growth processes. This study investigated the substrate preference of Streptomyces sp. F-3 using functional proteomic analysis. Streptomyces sp. F-3 exhibited varying degradation and utilization rates for different nitrogen source. The results indicated that the strain F-3 could not efficiently degrade intact globular proteins, but preferred to degrade peptone or protein hydrolysate, especially for waste-yeast. The strain F-3 could utilize waste-yeast to grow rapidly and produced a large amount of extracellular protein. The substrate-binding patterns of three S8 proteases secreted by Streptomyces sp. F-3 determined the nitrogen source degradation preference of the strain. In addition, the strain F-3 could secrete large amounts of β-glucanase and chitinase to utilize cell wall polysaccharides. Thus, waste-yeast, rich in peptone, β-glucan, and chitin, could be the superior substrate for culturing Streptomyces. This study not only broadens the application scenarios for waste-yeast, but also provides valuable insights for rapid and cost-effective industrial microbial cultivation.Key pointsThe substrate preference ofStreptomycessp. F-3 was analyzed by integrative omics.Structural omics revealed the hydrolysis specificity of S8 proteases from F-3.Waste-yeast served as the superior substrate for culturing Streptomyces.

Ergothioneine (EGT) is a rare amino acid with potent antioxidant and anti-inflammatory properties, with a wide range of applications in food, cosmetics, and medicine. In the present study, Aspergillus oryzae, a common edible fungus, was engineered as an optimal host for EGT production. Moreover, two endogenous genes involved in EGT biosynthesis were characterized. The homolog AoEgt1 was shown to be localized in the vacuoles, whereas the homolog AoEgt2 was found in the peroxisomes. Overexpression of EGT biosynthetic genes from different organisms enhanced EGT production, yielding 15.17 mg EGT/g of dry weight. Using glucose as the carbon source and supplementing methionine (Met) as a precursor further increased EGT production to 20.03 mg EGT/g of dry weight, constituting an eight-fold increase compared to the wild-type strain. This study discusses the successful construction of a high-yielding A. oryzae strain for EGT biosynthesis, providing a novel strategy for efficient EGT synthesis.Key points• Two newly described homologs, AoEgt1 and AoEgt2, were identified in A. oryzae.• AoEgt1 and AoEgt2 were found to contribute to EGT biosynthesis.• EGT production was significantly increased by overexpression of Egt1 and Egt2.• Glucose and Met supplementation in the medium increased EGT production.

Flavoprotein oxidases have found many biotechnological applications. For identifying and improving their characteristics, it is essential to have reliable and robust assay methodology available. The methodologies used to monitor their activity seem to be scattered in the literature and seem often selected based on convenience. Due to the diversity of reactions catalyzed by flavoprotein oxidases, it is virtually impossible to recommend a single activity assay. A literature analysis of 60 recent papers describing flavoprotein oxidases revealed that continuous spectrophotometric assays, in particular colorimetric assays, are the preferred choice, as they are facile, scalable and allow for better interpretation of data than discontinuous assays. Colorimetric assays typically rely on the extinction coefficient of a monitored chromogenic product, which can be highly variable depending on the experimental conditions. Therefore, it is important to determine the extinction coefficient under the specific experimental conditions used, rather than taking it directly from the literature. To provide a guideline and assist in standardization, this review describes the most commonly utilized activity assays for flavoprotein oxidases, along with their respective merits and limitations.Graphical Key points• Researchers should be more aware of limitations of activity assays.• Extinction coefficients should be determined for the appropriate experimental setup.• New robust activity assays are desired.

Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria during growth, mainly under stress conditions. OMV-based vaccines have played an important role in vaccination against Neisseria meningitidis serogroup B (MenB), stimulating research into novel approaches for developing more effective vaccines. OMVs released by the bacterium Neisseria lactamica have emerged as a promising platform for new vaccine development, especially as carriers in subunit vaccines. Despite their importance, some challenges remain in obtaining and purifying OMVs. The most commonly employed methods for OMV isolation and purification are ultracentrifugation (UC) and size exclusion chromatography (SEC). However, these techniques could present limitations for large-scale production and often result in low yields. This study investigated techniques such as tangential flow filtration (TFF), membrane chromatography, and mixed-mode (multimodal) chromatography as potential replacements for UC and SEC. Among the TFF methods evaluated, the sample obtained on the membrane with a 300-kDa cutoff showed a profile more similar to UC but with more than double the total protein recovery. Sartobind® Q membrane chromatography was ineffective for OMV purification, in the conditions evaluated, with a recovery of 8.7%. Conversely, multimodal Capto™ Adhere chromatography recovered 59.0%, while Capto™ Core 400 yielded a recovery of 72.0%, proving to be more effective for purification when analyzed by high-performance liquid chromatography (HPLC). Thus, combining TFF with a 300-kDa membrane followed by Capto™ Core 400 chromatography can be applied as strategy for large-scale applications offering high recovery and purity.Key points• Evaluation of TFF, membrane and multimodal chromatography techniques for OMV purification.• Improved Neisseria lactamica OMV yields combining TFF and multimodal chromatography.• A process for OMV purification from a non-pathogenic organism feasible to scale up.

Pet allergies are increasingly prevalent in developed nations, significantly affecting humans and strongly linked with asthma and rhinitis. Allergic reactions to cats and dogs affect 15.7% of Americans and 27.2% of Europeans, with sensitization rates to dog allergens reaching 56.0% in Denmark. Despite these concerns, dog ownership remains widespread, with 25% of European and 45.5% of US households owning at least one dog. With sensitization on the rise and current diagnostic and therapeutic approaches predominantly relying on inherently inconsistent allergen extracts derived from natural sources, recombinant allergen production offers a pathway to component-resolved diagnostics, improving specificity and reliability in allergy diagnosis. The present research explored, for the first time, the production of the allergen component glycoprotein Can f 6 in the eukaryotic expression system Pichia pastoris and compared its IgE antigenicity to recombinant Can f 6 (rCan f 6) variants produced in Escherichia coli. Yields were significantly increased by fusing Can f 6 with the maltose binding protein (MBP), resulting in a 1.8-fold increase in production in E. coli and a threefold increase in P. pastoris. Antigenicity analysis showed that N-glycosylation is not critical for folding or IgE recognition of Can f 6, making both systems equally suitable for producing the allergen. Notably, P. pastoris-produced MBP fused protein purified through cation exchange chromatography yielded a lower protein quantity. Still, it exhibited stronger IgE reactivity than the same protein purified using anion exchange chromatography.Graphical abstract

β-1,2-Glucans are physiologically important polymers for interactions such as symbiosis and pathogenesis between organisms and adaptation to environmental changes. However, rarity of β-1,2-glucans in nature limits exploration of related enzymes. Recently, many β-1,2-glucan-degrading enzymes have been found after identification of a novel phosphorylase acting on β-1,2-glucooligosaccharides. The expansion of the repertoire has reached revelation of the cyclization mechanism of cyclic β-1,2-glucan synthase and led to finding of new enzymes catalyzing cyclization of β-1,2-glucans in a manner different from cyclic β-1,2-glucan synthase. In this review, we mainly focus on newly found enzymes that catalyze cyclization of β-1,2-glucans along with existence of β-1,2-glucan-associated carbohydrates in nature and introduction of the repertoire of β-1,2-glucan-degrading enzymes.Key points• Newly found domain which cyclizes β-1,2-glucan created a new glycoside hydrolase family.• Cyclization is performed with a unique mechanism.• α-1,6-Cyclized β-1,2-glucan is produced by an enzyme in another newly found family.

Pollutant-derived risks to human and environmental health are exacerbated by slow natural attenuation rates, often driven by pollutant toxicity to microorganisms that can degrade them or limitations to the ability of microorganisms to metabolise them. This review explores mechanisms employed by bacteria to protect themselves from pollutant toxicity in the context of the evolution of pollutant-degrading abilities. The role of promiscuous enzymes in pollutant transformation is subsequently reviewed, highlighting the emergence of novel metabolic pathways and their transcriptional regulation in response to pollutant exposure, followed by the gene transcription regulation to optimise the cellular component synthesis for adaptation on the novel substrate. Additionally, we discuss epistatic interactions among mutations vital for this process both at macromolecular and at cellular levels. Finally, evolutionary constraints towards enhanced fitness in the context of pollutant degradation are considered, the constraints imposed by the epistasis from mutations on both enzyme level and cellular level, concluding with challenges and emerging opportunities to develop sustainable contaminated site remediation technologies.Key points•Pollutants can exert toxicity on cellular membrane, enzyme and gene transcription.•Bacteria can patch promiscuous enzymes into novel pathway to degrade pollutants.•The evolution trajectory is constrained by epistasis from mutations on enzyme and cellular level.

One strategy for CO2 mitigation is using photosynthetic microorganisms to sequester CO2 under high concentrations, such as in flue gases. While elevated CO2 levels generally promote growth, excessively high levels inhibit growth through uncertain mechanisms. This study investigated the physiology of the cyanobacterium Synechocystis sp. PCC 6803 under very high CO2 concentrations and yet stable pH around 7.5. The growth rate of the wild type (WT) at 200 µmol photons m−2 s−1 and a gas phase containing 30% CO2 was 2.7-fold lower compared to 4% CO2. Using a CRISPR interference mutant library, we identified genes that, when repressed, either enhanced or impaired growth under 30% or 4% CO2. Repression of genes involved in light harvesting (cpc and apc), photochemical electron transfer (cytM, psbJ, and petE), and several genes with little or unknown functions promoted growth under 30% CO2, while repression of key regulators of photosynthesis (pmgA) and CO2 capture and fixation (ccmR, cp12, and yfr1) increased growth inhibition under 30% CO2. Experiments confirmed that WT cells were more susceptible to light inhibition under 30% than under 4% CO2 and that a light-harvesting-impaired ΔcpcG mutant showed improved growth under 30% CO2 compared to the WT. These findings suggest that enhanced fitness under very high CO2 involves modifications in light harvesting, electron transfer, and carbon metabolism, and that the native regulatory machinery is insufficient, and in some cases obstructive, for optimal growth under 30% CO2. This genetic profiling provides potential targets for engineering cyanobacteria with improved photosynthetic efficiency and stress resilience for biotechnological applications.Key points• Synechocystis growth was inhibited under very high CO2.• Inhibition of growth under very high CO2 was light dependent.• Repression of photosynthesis genes improved growth under very high CO2.Graphical