In this study, a thermophilic cellulolytic filamentous fungus Thermothelomyces heterothallica was engineered for direct production of cellobionic acid (CBA) from alkali-pretreated wheat straw without the addition of external cellulases. The CBA bioproduction pathway was established through sequential disruption of genes involved in cellobiose and CBA catabolism, including eight β-glucosidases, one cellobiose/cellobionate phosphorylase, and two putative cellobionate transporters using a ribonucleoprotein (RNP)-based CRISPR-Cas9 system. The resulting T. heterothallica strain, TH11, was able to produce cellobionate from cellobiose and pretreated wheat straw at a high yield. Further optimization of fermentation pH using 50 mM citrate buffer (pH 6.0) increased the final cellobionate concentration. Fermentation temperature optimization showed that 43 °C accelerated substrate conversion without significantly affecting final titers. The engineered strain, TH11 produces 64 mM of cellobionate in 5 days. The cellulose conversion was about 88
Acute kidney injury (AKI) affects a considerable proportion of patients that represents a major challenge for clinical treatment. Gut microbiota metabolites have been reported to attenuate acute kidney injury (AKI), yet their underlying mechanisms remain largely elusive. The present study aimed to explore the protective mechanisms of these metabolites against AKI using. The targets of metabolites and AKI were obtained from public databases. The PPI network is utilized to identify the core targets. GO and KEGG enrichment analysis were employed to predict significant pathway and biological functions. The Gut-Metabolites-Targets-Pathway network (G-M-T-P) network was constructed to screen the core metabolites. Furthermore, molecular docking was applied to evaluate the binding affinities between the metabolites and their candidate targets. A total of 84 overlapping targets between gut microbiota metabolites and AKI were acquired. The beneficial effects of gut microbiota metabolites were associated with the regulation of inflammatory response and hypoxia. HIF-1 pathway was identified as the significant pathway mediating the effects of metabolites. STAT3, HIF1A, NFKB1, IL6 and AKT1 were identified as the core therapeutic targets of metabolites against AKI. The G-M-T-P network identified acetate, butyrate, propionate, and 3-indolepropionic acid as the core metabolites. The core metabolites exhibited strong affinity binding affinity for the core targets, suggesting their potential application in candidate drug development. The study highlights that the promising effects of gut microbiota metabolites in AKI treatment through modulating multi targets and pathways.
Microalgae (predominantly unicellular photosynthetic eukaryotes) has been recognized as a “protein bio-factory” because they may produce up to 70
The growing environmental impact of petroleum-based plastics has intensified the search for sustainable and biodegradable alternatives. Polyhydroxybutyrate (PHB), a microbial polyester from the polyhydroxyalkanoate (PHA) family, has emerged as a promising biopolymer due to its biodegradability, biocompatibility, and thermoplastic qualities that rival those of conventional polymers. Halophilic bacteria have attracted considerable attention among PHB-producing microorganisms because they thrive in hypersaline environments, enabling non-sterile cultivation, reducing contamination risks, and facilitating cost-effective downstream recovery by osmotic cell lysis. This review provides a comprehensive overview of recent improvements in PHB production by halophilic bacteria, covering physiological adaptations, metabolic pathways, substrate usage, fermentation techniques, and bioreactor optimisation. Particular emphasis is placed on the use of agro-industrial residues and waste-derived feedstocks as sustainable carbon sources to reduce production costs and increase circular bioeconomy results. Recent advances in downstream processing, such as green extraction technologies, metabolic engineering, CRISPR-based gene editing, and synthetic biology approaches to increasing PHB productivity, are critically reviewed. Additionally, developments in polymer modification, life cycle assessment, industrial scalability, regulatory frameworks, and potential applications in packaging, agriculture, and biomedical engineering are discussed. Despite significant progress, issues such as process economics, saline wastewater control, polymer brittleness, and large-scale commercialisation remain. Integrating halophilic biotechnology with waste valorisation, green recovery technologies, sophisticated metabolic engineering, and circular biorefinery concepts offers a promising strategy for developing economically and environmentally sustainable PHB production systems.
Chinese hamster ovary (CHO) cells represent the dominant host system for the production of recombinant therapeutic proteins. In recent decades, extensive research has focused on process/media optimization and cell line engineering to improve both the productivity and quality of biopharmaceutical proteins produced in CHO cells. Nevertheless, the inherent complexity of biological pathways and the heterogeneous cellular responses to different environmental conditions have posed substantial challenges to traditional methodologies. Recent advances in omics technologies have enabled comprehensive characterization of CHO cell physiology, providing multidimensional molecular and phenotypic insights that facilitate the enhancement of recombinant protein production. This review first summarizes the methodologies and advances in CHO omics research, including genomics, transcriptomics, proteomics, metabolomics, and epigenomics. It then examines contemporary approaches to integrate and analyze multi-omics data in CHO cells. The review further elucidates how these multi-omics datasets can be strategically applied across various developmental stages, including cell line selection, genetic engineering, expression vector design, and bioprocess optimization. Finally, we explore the transformative potential of integrating multi-omics with artificial intelligence and discuss promising future research directions in CHO cell studies. These emerging paradigms offer novel opportunities for data-driven cell engineering and bioprocess optimization in CHO-based biomanufacturing.
The marine protist Schizochytrium limacinum SR21 is a promising producer of docosahexaenoic acid (DHA) from short-chain carboxylic acids (SCCAs). Dark fermentation effluent (DFE) is an SCCA- and nutrient-rich substrate derived from biogenic residues and has previously been evaluated for microbial lipid production. However, its low carbon concentration limits volumetric productivity and causes substantial culture dilution when larger feed volumes are required. Membrane-based cell retention is an established process intensification strategy for low-concentration feed solutions, as it decouples hydraulic residence time from biomass residence time. However, the combination of DFE utilisation and cell retention for DHA production by S. limacinum has received little attention. In this study, repeated fed-batch and cell retention strategies were evaluated to overcome dilution limitations and enable intensified biomass and lipid production from DFE. Additional cultivations with defined SCCA mixtures showed preferential uptake of acetate and butyrate over propionate and lactate, which was also observed during DFE conversion. In repeated fed-batch operation, biomass output reached 7.5 g after 95 h. In contrast, a final biomass output of 33 g was achieved in cell retention mode, representing a 4.4-fold increase. This improvement was not associated with enhanced substrate conversion efficiency but resulted from retaining biomass while increasing DFE throughput. While applying cell retention, the specific palmitic acid concentration increased to 116 mg g⁻¹, while DHA remained comparatively stable at 57 mg g⁻¹. This resulted in a DHA titre of 1.9 g L⁻¹ compared with 0.24 g L⁻¹ in repeated fed-batch. Nitrogen and phosphate removal reached 58
Low-quality tobacco leaves generally exhibit undesirable sensory characteristics due to excessive macromolecule accumulation and insufficient aroma release. Fermentation with functional microorganisms to improve tobacco quality is a green and efficient strategy. This study employed volatile flavoromics and microbiomics to investigate the flavor basis and underlying microbial regulatory mechanism for quality improvement by Bacillus subtilis DB-15. Results showed that B. subtilis DB-15 fermentation significantly degraded starch, protein, and cellulose, and comprehensively enhanced sensory quality by increasing aroma and sweetness while reducing irritation and off-flavors. Subsequently, a total of 94 volatile compounds were identified by HS-SPME-GC-MS. B. subtilis DB-15 promoted the enrichment of esters and heterocycles, and inhibited harmful substances such as short-chain fatty acids and nicotine. Microbiomics analysis indicated that inoculated B. subtilis DB-15 dominated the bacterial community and reshaped its structure, whereas fungal community succession was mainly driven by water addition rather than Bacillus inoculation. Functional prediction suggested a higher relative abundance of bacterial pathways related to carbohydrate and amino acid metabolism in the Bacillus inoculation group, whereas pathways related to terpenoid and polyketide metabolism showed lower predicted abundances. Correlation network analysis showed that Bacillus was positively correlated with sweet and fruity aroma compounds but negatively correlated with irritant acids, suggesting a potential contribution to improved sensory coordination. This study reveals the mechanism of sensory improvement by B. subtilis DB-15 fermentation from dual-omics perspective, providing a reliable technical strategy for upgrading low-quality tobacco resources.
Enterobacterial infections being severe, with a high mortality rate, particularly affecting ICU patients, and 80
Abstract Alfalfa is a high-protein forage crop widely used in ruminant production, yet its ensiling is generally challenged by low water-soluble carbohydrate content and high buffering capacity. This study systematically evaluated the effects of microbial inoculants, cellulase, and sodium diacetate on fermentation quality, microbial succession, and predicted metabolic functions of alfalfa silage using fermentation analyses and 16S rRNA sequencing. Lactic acid was detected only in the microbial inoculant-treated silages on day 1, accompanied by a rapid decline in pH below 5.8 ( p < 0.05). After 60 days, all microbial inoculants except BSM lowered pH below 4.5 and reduced ammonia nitrogen (NH₃-N) ( p < 0.05), while promoting Lactiplantibacillus - or Pediococcus -dominated communities and suppressing Enterobacter and Serratia ( p < 0.05). Predicted functional profiles suggested lower lysine degradation and enhanced lysine biosynthesis. Among inoculants, KL yielded the most favorable profile, with the highest Lactiplantibacillus abundance (78.23%), lower gas loss, Chao1, Shannon, and NH₃-N (4.35% total nitrogen (TN)). Cellulase improved fermentation quality compared with the control and displayed the lowest fiber content, but showed slower early acidification, resulting in higher NH₃-N (6.46% TN), gas loss, and Enterobacter abundance (2.94%) after 60 days ( p < 0.05). Sodium diacetate followed a distinct pathway, producing acetic acid (1.32% dry matter (DM)) rather than lactic acid on day 1 and reducing pH to 5.58. After 60 days, sodium diacetate produced well-preserved silage with a lower pH (4.28), NH₃-N (3.72% TN), and gas loss, higher lactic acid concentration (6.93% DM) ( p < 0.05), and Lactiplantibacillus -dominated bacterial community. These findings highlight that silage additives govern fermentation outcomes through distinct mechanisms, with the KL inoculant and sodium diacetate providing the most effective preservation strategies in this study. Graphical abstract
Tomato pomace, a byproduct of tomato processing industry, can be utilized through fermentation to produce single cell protein (SCP). In this study, fermentation parameters were optimized in a 3-L bioreactor using a full factorial design (FFD), to which a second order polynomial regression model was subsequently fitted to locate the optimum via response surface analysis. While pH, temperature, agitation speed and airflow rate were held constant, the effects of solid load, inoculum volume, and fermentation period were analyzed using a 3 × 3 × 5 experimental design. The resulting biomass was harvested, and its protein content was analyzed. Optimal conditions were identified as a 10.35
As a widely used anticoagulant, heparin is industrially produced chiefly via animal tissue extraction, which suffers from unstable supply and potential safety hazards. Heparosan shares a similar polysaccharide backbone with heparin and can be converted into heparin under mild enzymatic catalysis. In addition, heparosan exhibits favorable biocompatibility and non-immunogenicity, rendering its efficient, eco-friendly biosynthesis essential. In this study, we systematically engineered Corynebacterium glutamicum, a Generally Recognized as Safe (GRAS) microorganism, to synthesize heparosan via two complementary strategies. First, genome-scale modification was implemented to stably upregulate genes ugd, glmS, and ndk. The heparosan titer of recombinant strain Cg24 increased from 226.37 to 595.39 mg/L. Second, translation-level fine-tuning was implemented to modulate expression of individual genes within the kfiB-kfiC-kfiA cassette by constructing a high-coverage random ribosome binding site (RBS) library, which further lifted heparosan titer to 1161.37 mg/L. In fed-batch fermentation using a 5 L bioreactor with a two-stage growth-production regulation strategy, the recombinant strain Cg24-11 produced 5.36 g/L of heparosan, demonstrating its great potential for efficient heparosan biosynthesis. This combined modification strategy also provides valuable references for constructing high-efficiency cell factories for other complex compounds.
Traditional soft cheeses are susceptible to microbial contamination, particularly when unsterilized flavoring additives are used during production or retail handling. This study aimed to quantify and molecularly characterize the yeast flora associated with spiced Karish cheese, a traditional Egyptian fresh cheese supplemented with peppers and olives. Yeast counts ranged from 2.25 to 2.99 log10 CFU/g, while molds were detected in 60
Nitrogen deprivation (N−) is widely used to induce lipid accumulation in microalgae. However, its impact on the relationship between lipid accumulation and thermochemical behavior remains poorly understood. In this study, a comparative experimental approach was used to evaluate the effects of nitrogen availability on growth kinetics, lipid accumulation, and pyrolysis-derived volatile compounds in Chlorella vulgaris, Scenedesmus obliquus, and Nannochloropsis oculata. Under nitrogen˗replete conditions (N +), all species showed higher growth rates, whereas N− reduced proliferation but increased lipid content by 5.0-fold in C. vulgaris, 2.4-fold in S. obliquus, and 1.8-fold in N. oculata. Py-GC/MS analysis revealed that N− shifted the pyrolysis profile toward lipid˗derived compounds, particularly C16˗C18 fatty acids, long˗chain alcohols, and hydrocarbons. In C. vulgaris, oleic acid (22.3
The coexistence of organic dyes and heavy metals in wastewater requires efficient and sustainable adsorbents capable of simultaneous contaminant removal. In this study, pecan shell–based activated carbons (PSC-0–PSC-3) were prepared by KOH activation using different impregnation ratios; their physicochemical properties and adsorption performance toward methylene blue (MB) and Cd(II) were systematically investigated under single- and binary-component systems. Among the prepared adsorbents, PSC-3 exhibited the highest BET specific surface area (2857 m2 g−1) and total pore volume (1.61 cm3 g−1). MB adsorption was best described by the Freundlich isotherm model (R2 = 0.998), indicating heterogeneous adsorption behavior. The Langmuir model estimated a maximum adsorption capacity of 529.5 mg g−1. Under binary-component conditions, PSC-3 achieved nearly complete MB removal (≈ 100
Green synthesis of zinc oxide nanoparticles (ZnO-NPs) using Mesembryanthemum crystallinum L. (M. crystallinum), a halophytic plant adapted to saline environments of North Sinai, Egypt, offers a sustainable nanofabrication strategy with inherent therapeutic potential. High-resolution LC-ESI-QTOF-MS metabolomic profiling identified 13 major phytochemicals in the ethanolic leaf extract, dominated by citramalate (peak area 31,601), L-phenylalanine (30,377), and stress-responsive organic acids, reflecting the plant’s halophytic adaptation. These redox-active compounds templated the biosynthesis of crystalline ZnO-NPs under ambient aqueous conditions, yielding spherical to quasi-spherical nanoparticles (4.4–12.2 nm primary size; 40–150 nm aggregates) with a hexagonal wurtzite structure, as confirmed by XRD and SAED. FTIR and EDX analyses verified surface functionalization by phytochemical capping agents (polyphenols, organic acids), while SEM revealed characteristic aggregation with rough surface morphology indicative of biomolecular adsorption. Biosynthesized ZnO-NPs exhibited selective cytotoxicity against human malignant melanoma A375 cells (IC₅₀ = 100.55 ± 8.6 µg/mL) with four-fold lower toxicity toward normal human skin fibroblasts (IC₅₀ = 406.01 ± 35.2 µg/mL; selectivity index ≈ 4.0). Mechanistic investigations demonstrated that nanoparticle internalization triggered robust ROS generation ( 3.8 × 10⁴ fluorescence units), mitochondrial membrane depolarization, and mixed apoptotic/necrotic cell death (40
In recent years, the production of microbial biosurfactants, increasingly used in various industries and agriculture, from low-cost renewable substrates is intensively studied. Large volumes of food waste, in particular waste cooking oil, are promising substrates for the production of biosurfactants on an industrial scale. However, studies on the ability of Rhodococcus bacteria, well-known biosurfactant producers, to grow on this substrate and the functional characteristics of synthesized biosurfactants are still scarce. In this study, the possibility of improving biosurfactant production by Rhodococcus ruber IEGM 231 grown on waste cooking oil was investigated using multifactor analysis and response surface methodology (RSM). Using standardized parameters for the concentrations of carbon and nitrogen sources in the fermentation medium, a Pareto diagram was constructed, which shows a direct dependence of the crude biosurfactant yield on the oil concentration, while the concentrations of sugar and inorganic nitrogen salts were found insignificant. Using RSM, the optimal ratio of the medium parameters was determined for the maximum yield of crude biosurfactants: oil (+ 1), sugar (0), NH4- and NO3-containing salts (− 1) in the following concentrations: 5.0 vol
3’-Sialyllactose (3’-SL), the simplest sialylated human milk oligosaccharide (HMO), is widely incorporated into infant formula due to its roles in shaping the gut microbiota, inhibiting pathogen adhesion, and supporting neurodevelopment. In this study, we developed an efficient microbial cell factory for 3’-SL production by enhancing carbon utilization and CTP regeneration. Following the identification of a highly active α-2,3-sialyltransferase, metabolic flux was redirected through pfkA deletion and fbaA overexpression. The precursor pool was strengthened by optimizing the expression of key enzymes involved in UDP-GlcNAc and CMP-Neu5Ac biosynthesis. To further improve pathway balance and stability, promoter engineering was applied to optimize the expression of four key genes (neuA, neuB, neuC and ST). The optimized multigene cassette was subsequently integrated into the chromosome using a one-step multicopy integration strategy, generating a plasmid-free and antibiotic marker-free production strain. The resulting strain achieved 3’-SL titers of 7.75 g L−1 in shake flasks and 102.18 g L−1 in a 5-L fed-batch bioreactor, representing, to the best of our knowledge, the highest reported level to date. This work demonstrates a robust strategy combining metabolic rewiring, adaptive gene expression tuning and multicopy genome integration, providing a versatile platform for the sustainable production of 3’-SL and other high-value HMOs.
With rising global demand for high-quality food crops, soybean production is constrained by an inherent yield-quality trade-off that conventional practices cannot easily overcome. Zinc oxide nanomaterials (ZnO NMs) show great potential for crop improvement, but their molecular mechanism underlying soybean quality regulation remains unclear. This study investigated the effects of ZnO quantum dots (ZnO QDs, 5.56 nm) and ZnO nanoparticles (ZnO NPs, 29.68 nm) on soybean growth and quality via root and foliar application with different concentrations (0, 5, 10, 20, 50 and 100 mg/kg). Among these experimental groups, root application of 50 mg/kg ZnO QDs (R-QD-50) was selected as the optimal combination based on regular analyses and used for integrated transcriptomic, proteomic and metabolomic analyses. The results showed that ZnO NMs significantly promoted soybean growth, yield and quality in ZnO NMs type, application mode and concentration-dependent manner. Multi-omics integration identified three common pathways: taurine and hypotaurine metabolism, butanoate metabolism, and phenylpropanoid biosynthesis. Among them, only phenylpropanoid biosynthesis formed a complete transcriptome-proteome-metabolome regulatory chain. Key genes phenylalanine ammonia lyase (PAL) and cinnamic acid-4-hydroxylase (C4H) were significantly upregulated, the related synthases increased by 35
Furfural and 5-hydroxymethylfurfural (HMF) are two major lignocellulosic growth inhibitors that hinder microbial growth and fermentation of lignocellulosic hydrolysate for lactic acid production. In this study, we employed adaptive laboratory evolution (ALE) to enhance the tolerance of Lactiplantibacillus plantarum JGR2, a strain previously isolated in our lab, to furfural and HMF. The adapted strains demonstrated significantly improved growth in the presence of these inhibitors compared to the parental strains. Whole-genome resequencing revealed multiple mutations including high-impact non-conservative mutations in genes encoding DNA recombination and repair protein (RecF, lp_0005), flavin prenyltransferase (UbiX lp_0271), and oligo-1,6-glucosidase (lp_0189). Transcriptomic analysis indicated that adaptation elicited more pronounced differential gene expression compared to acute inhibitor exposure. Upon furfural exposure, the furfural-adapted isolate showed fewer differentially expressed genes than the parental strain, indicating a possible shift in the transcriptomic profile as a possible mechanism of furfural adaptation. Mechanistic investigation revealed that the adapted isolates reduce furfural into the less toxic furfuryl alcohol, suggesting a key detoxification mechanism. Notably, lp_3051 (dhaT, 1,3-propanediol dehydrogenase) encoding furfural reductase activity was upregulated in both furfural- and HMF-adapted isolates. Membrane fatty acid analysis revealed increased unsaturated fatty acids and cyclopropane fatty acids in adapted strains. Finally, the adapted strains exhibited improved growth in rice straw hydrolysate and produced significantly higher relative lactic acid yields compared to the parental strain, thus demonstrating improved bioproduction under inhibitor-rich conditions. This study not only provides a comprehensive understanding of L. plantarum’s response to lignocellulosic inhibitors, but also yields evolved bacterial candidates for further scientific and industrial exploration.
Raman spectroscopy is increasingly used as a process analytical technology (PAT) for real-time monitoring of bioprocesses. However, chemometric models developed using high-throughput (HT) mini-bioreactor systems often show limited predictive performance when applied to larger-scale processes, reflecting an out-of-distribution (OOD) challenge in cross-scale model transfer. In this study, we investigated whether variable-specific data preprocessing strategies can improve the cross-scale prediction performance of Raman chemometric models calibrated using HT cell culture data. Multiple preprocessing approaches were systematically evaluated for key cell culture target outputs, and the optimal preprocessing pipeline for each output was selected based on its ability to minimize cross-scale prediction error. The optimized variable-specific preprocessing pipelines reduced cross-scale prediction errors by 14.0–56.1
