Search Results (196)

Komagataella phaffii (formerly Pichia pastoris) is a prominent platform for recombinant protein production, yet secretion efficiency often remains a critical bottleneck. In this study, we validated three candidate genes—WHI3, CLPP, and PMP20—previously identified through genome-wide CRISPR activation screening, for their potential to enhance heterologous protein secretion. Overexpression of these factors under the control of the methanol-inducible AOX1 promoter increased the secretion of human serum albumin (HSA), with WHI3 and CLPP yielding improvements of 18.3% and 17.9%, respectively. Furthermore, applying this strategy to human lactoferrin (hLF) revealed that WHI3 overexpression robustly enhanced hLF secretion by approximately 70%. Comparative analysis of different promoters (AOX1, GAP, and CAT) indicated that the AOX1 promoter remains the most effective driver for these enhancers, suggesting a threshold-dependent regulatory mechanism. These results demonstrate the protein-dependent nature of secretion optimization and identify WHI3, CLPP, and PMP20 as novel, effective co-expression factors for improving recombinant protein yields in K. phaffii. Full article

Moist chilling is widely used to overcome seed dormancy in zoysiagrass (Zoysia japonica Steud.), but the coordinated physiological and molecular basis remains unclear. Here, freshly matured seeds were subjected to moist chilling at 4 °C in darkness for 0 (Control), 1 (CS1), 2 (CS2), 3 (CS3), or 4 weeks (CS4) and then transferred to germination conditions (30/20 °C, day/night). Prolonged moist chilling progressively improved dormancy release: final germination percentage increased from 40.5% (Control) to 73.5% (CS4), accompanied by a higher germination index and earlier, faster cumulative germination dynamics. Moist chilling also enhanced early seedling vigor, with stronger treatment differentiation in root elongation than in shoot growth. Physiologically, abscisic acid (ABA) content declined while gibberellic acid (GA) content increased, resulting in an elevated GA/ABA ratio with prolonged chilling. Metabolic activation was evidenced by increased α-amylase activity, greater soluble sugar and soluble protein accumulation, and stimulated oxygen uptake. In addition, CAT, SOD, and POD activities were enhanced under prolonged moist chilling, whereas H₂O₂ levels remained relatively stable, suggesting that redox adjustment during dormancy release was characterized by strengthened antioxidant buffering rather than pronounced oxidative accumulation. qRT-PCR supported a mechanistic transition from dormancy maintenance to germination execution, showing moist chilling-associated regulation of ABA/GA metabolism and signaling genes (e.g., NCED, CYP707A, ABI3/ABI5, and GA20ox) and downstream metabolic modules (e.g., GAMYB, AMY, ISA, INV, and HXK1), together with concurrent modulation of respiration- and ROS-related markers (e.g., AOX1a, RBOH, and CAT). Correlation analysis linked germination performance most strongly with α-amylase activity, oxygen uptake, and the GA/ABA ratio. Collectively, our data support a working model in which moist chilling rebalances the ABA–GA gate and activates downstream metabolic and redox adjustment modules to promote dormancy release and improve germination performance in zoysiagrass, providing practical markers for optimizing seed establishment through moist chilling treatment. Full article

Enhancing anthocyanin accumulation in red-leaf lettuce grown in plant factories often incurs yield penalties. Here we propose a two-stage screening-to-optimization framework integrated with mechanistic modeling to resolve this tradeoff. In Stage 1, comparative experiments confirmed that UV-A is more compatible with growth and pigmentation than UV-B, and identified ‘Lollo Rosso’ as a highly responsive cultivar. In Stage 2, optimization experiments showed that L6D6 (6 h day⁻¹ for 6 days) increased the total anthocyanin per plant by 19.9% while maintaining fresh weight. Motivated by observed nonlinear phenomena including biomass overcompensation, circadian disruption under night irradiation, and ontogeny-dependent vulnerability, we developed a six-state ordinary differential equation (ODE) model that integrates reactive oxygen species (ROS) dynamics with stress damage–repair processes. A key innovation is the explicit representation of carbon competition between growth and antioxidant defense, where AOX synthesis consumes carbon from the buffer pool, creating a physiologically meaningful growth–defense tradeoff supported by the Growth-Differentiation Balance Hypothesis. The model achieved high accuracy in an independent validation set that included extreme doses (errors ≤ 10.6%, with 11 of 12 metrics < 10%), supporting the physiological necessity of the introduced mechanisms. Global optimization based on the calibrated model predicted that 9 h day⁻¹ for 4 days is the theoretical optimum, potentially increasing total anthocyanin by 38.3% with minimal fresh-weight reduction (−2.4%), substantially outperforming the best experimental treatment. This quantitative mechanistic framework provides a scientific basis for designing precise stress-light recipes in controlled-environment agriculture. Full article

Bovine chymosin is key for cheese production, yet its traditional sourcing is unsustainable. While microbial and plant-based alternatives exist, they often cause non-specific proteolysis, leading to bitter flavors in cheese. This study aims to develop a high-yield, methanol-independent platform for recombinant bovine chymosin production by engineering the expression system of Komagataella phaffii through multi-factorial optimization. Initially, the native bovine prochymosin gene (pcw) was codon-optimized (pcm14) and cloned, along with an mCherry-tag construct (clpcm14), into inducible vector pPIC9 for expression in Komagataella phaffii GS115. Screening identified the fusion-tagged strain clp2-91 as the highest producer. Subsequently, the inducible AOX1 promoter in the previously selected clp2-91 strain was replaced with a constitutive GAP promoter, yielding engineered strain GH1. Cultivated in a 3L fermenter, GH1 exhibited a volumetric productivity of 105.03 SU/(mL·h), twice that of inducible strain clp2-91 (53.59 SU/(mL·h)). The further optimization of fermentation conditions (pH 4.0, glucose as carbon source, fed-batch mode) boosted the enzyme activity of GH1 to 12,000 SU/mL. The recombinant chymosin exhibited enzymatic properties similar to those of the native enzyme and, importantly, demonstrated a broader pH stability (pH 2.0–6.0). This study demonstrates an efficient strategy for chymosin expression in K. phaffii, offering insights that may support the future development and optimization of heterologous protein production in this yeast. Full article

Background/Objectives: Nutraceuticals, including short-chain fatty acids (SCFAs) and antioxidants (AOXs), are nutrient-derived bioactive compounds considered as potential treatments for metabolic-associated steatotic liver disease (MASLD). However, in vitro studies of their effects are limited by inconsistent experimental conditions, including differences in cell lines, methods of steatosis induction, and culture media, and by reliance on qualitative rather than quantitative assessments. Here, we systematically evaluate the anti-steatotic potential of eight commonly used nutraceuticals—three SCFAs (butyrate, acetate, and propionate) and five AOXs (resveratrol, curcumin, berberine, chlorogenic acid, and vitamin E)—using a standardized in vitro approach. Methods: Following a systematic literature review to identify common experimental conditions, we developed an assay to validate steatosis induction and quantified the effects of the nutraceuticals. For our studies we used the HepG2 liver cancer cell line and the Fa2N-4 immortalized hepatocyte cell line. Steatosis was modeled by stimulating cells with free fatty acids and fructose for 48 h. Nutraceuticals were added either concurrently with steatotic stimulation, to assess preventive effects, or after 24 h to assess therapeutic effects. Anti-steatotic drugs (resmetirom, semaglutide, obeticholic acid, and a DGAT2 inhibitor) were included as positive controls. Intracellular triglyceride levels were measured to quantify steatosis. Results: A systematic review of 46 studies revealed large differences in culture conditions, steatosis induction, and nutraceutical assessment. In our experiments, most nutraceuticals did not reduce intracellular triglycerides, with the exception of vitamin E. Surprisingly, butyrate, berberine, and curcumin increased triglyceride accumulation. Resmetirom was the only drug that significantly decreased triglycerides, while obeticholic acid, semaglutide, and the DGAT2 inhibitor showed minimal or inconsistent effects. Fa2N-4 cells were generally more sensitive than HepG2 cells, showing larger absolute changes in triglyceride levels in response to both nutraceuticals and resmetirom. Conclusions: We established a standardized in vitro assay to evaluate the anti-steatotic potential of nutraceuticals. Using this system, we found that SCFAs and AOXs did not consistently reduce intracellular triglycerides, highlighting the need for quantitative assessments and careful validation when studying anti-steatotic interventions in vitro. Full article

BslA (Biofilm surface layer protein A), a highly hydrophobic lipoprotein from Bacillus spp., self-assembles at fluid interfaces to form a crystalline film that reduces surface tension. In this study, we selected Pichia pastoris as a eukaryotic system for expressing recombinant BslA identified in Bacillus paralicheniformis BL-1. The secretory expression of recombinant BslA in the P. pastoris GS115 strain under the AOX1 promoter was confirmed in shake-flask cultivation. Next, two fed-batch fermentation strategies, constant dissolved oxygen strategy (DO-stat) and oxygen-limited fed-batch (OLFB) strategy, in a 5 L scale, were compared. The DO-stat process led to late-stage cell death and product degradation, limiting yields. Switching to the OLFB process by removing the glycerol feeding phase mitigated this issue, allowing extended fermentation and increasing the final recombinant BslA concentration to 657 mg/L. This study establishes P. pastoris with an OLFB strategy as an effective system for secreting recombinant BslA protein, providing a basis for future industrial-scale production. Full article

The sensory quality of pork constitutes a complex phenotype that arises from the interplay between genetic factors and environmental conditions. As a local pig breed in China, Tibetan pigs (TPs) are known for their high-quality meat. However, their slow growth rate and low production efficiency limit their large-scale breeding. We have used Duroc as a hybrid sire to improve TP. Our study found that TPs have higher intramuscular fat content and higher levels of monounsaturated fatty acids. Duroc × Tibetan crossbred pigs (DZs) not only retain the paternal high productivity but also inherit the superior meat quality of the maternal parent. Transcriptome analysis identified IL6, GPX1, GPX3, AOX1, ALDH7A1, PTGS2, NFKBIA, ADIPOQ and PPARG as being involved in affecting meat quality. Metabolomic analysis found that betaine, carnosine, L-carnitine, and lysophosphatidylcholine were important components that affect meat quality. Joint analysis further reveals that the expression of ATF4, DGKB, GNMT, and ADSL genes is closely related to arachidonic acid, lysophosphatidylcholine, betaines, and hypoxanthine, ultimately affecting the quality of the meat. By comprehensively analyzing the carcass and meat quality traits, genes and metabolites affecting meat quality traits, this study provides new evidence for improving pork quality and guiding breeding strategies. Full article

The study evaluated the feasibility of using the activated sludge process to treat real wastewater from used oil installations containing petroleum hydrocarbons, boron (B), and adsorbable organic halides (AOX). The aim was to determine the maximum ratio of this wastewater that could be added to the influent without impairing treatment efficiency. Tested shares ranged from 0.50% to 1.90%. An initial 1.30% of the tested share caused process instability, reflected in the elevated total nitrogen (TN) levels in treated wastewater. After reducing the share to 0.50%, an adaptation of the activated sludge was observed, manifested by a decrease in TN concentration to below 15.0 mg N/L. For the most favorable share of 1.60% (0.38 ± 0.10 kg_BOD5/kg_MLSS d, 0.51 ± 0.14 kg_COD/kg_MLSS d), the removal efficiencies of chemical oxygen demand (COD), biochemical oxygen demand (BOD₅), TN, and total phosphorus (TP) were 95.0% ± 1.5, 99.1% ± 0.2, 89.3% ± 2.7, and 94.0% ± 5.0, respectively. Increasing the share to 1.90% decreased treatment efficiency and exceedances of COD, BOD₅, TN, and TP occurred. At this ratio, an increase in ammonium nitrogen (NH4⁺-N) and TN concentrations was observed, indicating the inhibition of nitrification. However, the average concentrations of mineral oil index, AOX and B in the treated wastewater remained within permissible levels throughout the study. Full article

Itaconic acid (IA) is an important bio-based platform chemical produced via submerged fermentation by the filamentous Ascomycete Aspergillus terreus. In this study, we examined the impact of initial phosphate concentration on IA production from D-glucose and D-xylose in optimized, manganese-limited fermentations. Nine phosphate concentrations ranging from 0.04 to 4 g L⁻¹ were tested, and representative low (0.04 g L⁻¹), optimal (0.1 g L⁻¹), and high (0.8 g L⁻¹) conditions were analyzed in detail in controlled, 6 L scale bioreactors. Phosphate availability primarily influenced biomass formation and the biomass-to-product ratio rather than directly affecting IA accumulation. Both lower- and higher-than-optimal phosphate concentrations decreased the volumetric and specific IA yields, while the highest productivity was observed at 0.1 g L⁻¹. Expression of the aoxA gene, encoding the cyanide-resistant alternative oxidase (AOX), and AOX enzymatic activity were inversely correlated with extracellular phosphate concentration, consistent with a role in redox homeostasis under phosphate-limited conditions. In contrast, total respiration rates and pellet-type morphology remained unaffected. These findings indicate that phosphate acts mainly as a secondary modulator of IA fermentation performance through its influence on biomass formation, whereas other metabolic constraints play a more dominant role in controlling IA overflow in A. terreus. Full article

Thanks to both endogenous and exogenous antioxidants (AOs), the antioxidant defense system ensures redox homeostasis, which is crucial for protecting the body from oxidative stress and maintaining overall health. The food industry also exploits the antioxidant properties to prevent or delay the oxidation of other molecules during processing and storage. There are many classical methods for assessing antioxidant capacity/activity, which are based on mechanisms such as hydrogen atom transfer (HAT), single electron transfer (SET), electron transfer with proton conjugation (HAT/SET mixed mode assays) or the chelation of selected transition metal ions (e.g., Fe²⁺ or Cu¹⁺). The antioxidant capacity (AOxC) index value can be expressed in terms of standard AOs (e.g., Trolox or ascorbic acid) equivalents, enabling different products to be compared. However, there is currently no standardized method for measuring AOxC. Nanoparticle sensors offer a new approach to assessing antioxidant status and can be used to analyze environmental samples, plant extracts, foodstuffs, dietary supplements and clinical samples. This review summarizes the available information on nanoparticle sensors as tools for assessing antioxidant status. Particular attention has been paid to nanoparticles (with a size of less than 100 nm), including silver (AgNPs), gold (AuNPs), cerium oxide (CeONPs) and other metal oxide nanoparticles, as well as nanozymes. Nanozymes belong to an advanced class of nanomaterials that mimic natural enzymes due to their catalytic properties and constitute a novel signal transduction strategy in colorimetric and absorption sensors based on the localized surface plasmon resonance (LSPR) band. Other potential AOxC sensors include quantum dots (QDs, <10 nm), which are particularly useful for the sensitive detection of specific antioxidants (e.g., GSH, AA and baicalein) and can achieve very good limits of detection (LOD). QDs and metallic nanoparticles (MNPs) operate on different principles to evaluate AOxC. MNPs rely on optical changes resulting from LSPR, which are monitored as changes in color or absorbance during synthesis, growth or aggregation. QDs, on the other hand, primarily utilize changes in fluorescence. This review aims to demonstrate that, thanks to its simplicity, speed, small sample volumes and relatively inexpensive instrumentation, nanoparticle-based AOxC assessment is a useful alternative to classical approaches and can be tailored to the desired aim and analytes. Full article

Stenocereus fruits are appreciated for their flavor and color, and their cultivation is highly sustainable, as they grow in arid zones without the need for fertilizers or agrochemicals. However, their nutritional and bioactive composition remains underexplored. This study evaluated the physicochemical and nutritional properties, bioactive compound content, and antioxidant capacity (AOX) of Stenocereus thurberi (red, white, purple, and orange), Stenocereus martinezzi (red), and Stenocereus gummosus (red). All fruits exhibited low total soluble solids (12.6 ± 0.2–14.7 ± 0.3 °Brix), acidity (0.81 ± 0.03–1.12 ± 0.03%), and moderate dietary fiber content (3.71 ± 0.05–4.86 ± 0.09%). S. martinezzi stood out for its high levels of betalains (33.7 ± 0.65 mg/100 g_fw), vitamin E (84.7 ± 0.2 µg/100 g_fw), and vitamin C (147.6 ± 11.4 mg/100 g_fw). At the same time, potassium, magnesium, and calcium were the predominant minerals in all samples. S. gummosus showed the highest total soluble phenols (120.6 ± 2.2 mg/100 g_fw) and was also notable for its flavonoid content. Flavonoids, hydroxycinnamic, and hydroxybenzoic acids were detected in all fruits. Red fruits had the highest AOX levels, followed by white, orange, and purple varieties. In conclusion, these fruits are nutritious, low in sugars, and rich in bioactive compounds, suggesting their potential as functional foods, particularly beneficial for individuals with chronic degenerative diseases. Full article

Wheat bran (WB) is a rich source of phenolic compounds (PCs) with antioxidant capacity (AOX). Approximately 90% of these PCs are bound to the cell wall matrix, which limits their bioavailability. Fermentation with Saccharomyces cerevisiae is an effective strategy to release these bound phenolics. This study aimed to evaluate the effect of fermentation on the release of bound PCs in WB and their AOX during an in vitro digestion system. WB was fermented with Saccharomyces cerevisiae for 2, 4, and 6 days and subsequently subjected to simulated digestion. Free PCs were extracted with methanol, while bound PCs were obtained through alkaline hydrolysis. Total PCs were quantified using the Folin–Ciocalteu method, and AOX was assessed through DPPH, TEAC, and FRAP assays. The content of bound PCs significantly increased after fermentation (p < 0.05): 30.24 ± 0.06 mg GAE/g (day 2), 27.18 ± 0.40 mg GAE/g (day 4), and 28.41 ± 0.40 mg GAE/g (day 6), compared with unfermented WB (7.7 ± 0.21 mg GAE/g) (p < 0.05). AOX was notably enhanced; DPPH reached its peak on day 4 (47.38 ± 0.07 µmol TE/g) (p < 0.05). TEAC was highest on day 2 (26.20 ± 0.43 µmol TE/g) compared with the control (20.14 ± 0.22 µmol TE/g) (p < 0.05) and FRAP showed a slight improvement on day 6 (57.38 ± 0.10 µmol TE/g) relative to the control (56.22 ± 0.13 µmol TE/g) (p < 0.05). Fermentation with Saccharomyces cerevisiae promotes the release of bound PCs in WB and enhances its AOX, highlighting its potential as a functional food ingredient. Full article

Tris(2-chloroethyl) phosphate (TCEP), as an organophosphate contaminant, poses a significant threat to the growth and development of plants, especially roots. This study aimed to clarify the mechanisms of TCEP’s toxicity and damage to root systems, as well as the mechanisms of its damage to the respiration and energy metabolism of alfalfa root cells. The results showed that TCEP obviously affected the root length, root surface area, root volume, and root diameter of alfalfa. With increasing stress intensity, the total mitochondrial respiration rate and Cytochrome C Oxidase (COX) pathway respiration rate progressively declined, while the Alternative Oxidase (AOX) pathway respiration rate and its proportion of total respiration gradually rose. In addition, adenosine triphosphate (ATP) content and root vigor were significantly reduced. Moreover, with an increase in TCEP concentration, root superoxide anion radical content in alfalfa root cells was significantly elevated, while superoxide dismutase (SOD) and catalase (CAT) activities were significantly lowered, and ascorbate peroxidase (APX) and peroxidase (POD) activities were significantly enhanced. The present study indicated that respiration was disrupted, causing a lack of ATP in root cells under TCEP. Both the overproduction of reactive oxygen species (ROS) from the mitochondrial respiratory electron transport chain (mECT) and the deficiency of ROS-scavenging enzymes caused ROS accumulation, which led to the destruction of the cell membrane structure and exacerbated the disruption of the respiratory metabolism. The disruption of the conversion and reuse of energy by TCEP affected root growth and development. Full article

A novel diamine oxidase (DAO) was discovered in the bacterium Glutamicibacter halophytocola (DAO-GH). The gene of DAO-GH was integrated into the genome of the yeast Komagataella phaffii and recombinantly produced under control of the methanol-inducible AOX1 promoter in a bioreactor cultivation. A high DAO activity of 70.2 ± 5.2 µkat/L_culture (5.25 ± 0.22 µkat/g_protein) was yielded after 90 h of cultivation. The DAO-GH was partially purified by the polyethyleneimine precipitation of nucleic acids, fractionated ammonium sulfate precipitation and hydrophobic interaction chromatography, resulting in a specific DAO activity of 19.7 µkat/g_Protein. The DAO-GH was then biochemically investigated regarding its potential for histamine and tyramine degradation in fermented foods and the human small intestine. Interestingly, the DAO-GH showed activity even at a low pH of 5 and low temperature of 6 °C. Both histamine and tyramine were effectively degraded and DAO-GH showed especially very high affinity towards tyramine (K_m of 0.009 mM). The DAO-GH was shown to be capable of degrading around 20% of the initially applied histamine in tuna paste (pH 5.6) at 5 °C within 24 h and completely degraded the histamine in a simulated intestinal fluid within 1.5 h in bioconversion experiments. The DAO-GH was spray-dried for the production of a storable enzyme preparation. Only around 17% of activity were lost in this process and the DAO-GH remained stable at room temperature for at least 3 months. The discovery of this DAO with its very advantageous biochemical properties allows the preparation of histamine-reduced or -free fermented foods by a simple enzymatic treatment or the treatment of histamine intolerance symptoms as a dietary supplement or medicine. Full article

The interaction of disinfectants with bromide/iodide ions and natural organic matter (NOM) generates brominated/iodinated disinfection byproducts (Br/I-DBPs), known for their heightened cytotoxicity and genotoxicity relative to chlorinated DBPs. This study investigated Br/I-DBP formation during sodium hypochlorite (NaClO) disinfection of lignin-containing synthetic water by quantifying adsorbable organic halogens (AOX) and trihalomethanes (THMs). Disinfection of bromide-containing water yielded a bromide ion (Br⁻) to adsorbable organic bromine (AOBr) conversion rate of approximately 60%, with bromine within THMs accounting for about 30% of the total AOBr, indicating significant brominated DBP formation where THMs represent a major fraction. Conversely, iodide ion (I⁻) conversion to adsorbable organic iodine (AOI) is minimal, suggesting negligible iodinated DBP formation under NaClO disinfection. Examination of key parameters revealed that brominated THM (Br-THM) formation decreased with increasing lignin concentration, while iodinated THM (I-THM) formation increased. The effect of chlorine dose differed: Br-THM formation exhibited an initial increase followed by a decrease at higher doses, whereas I-THM formation consistently decreased. Both Br-THM and I-THM formation increased with higher bromide or iodide ion concentration and with increasing pH. These results highlight the distinct pathways and influencing factors governing brominated versus iodinated DBP formation in lignin-containing waters disinfected with hypochlorite. Full article