Biotechnology Letters最新文献

To investigate the effects of fungal volatile organic compounds (FVOCs) on the mycelial growth of Ganoderma lucidum, and to elucidate the molecular mechanisms underlying the growth-promoting effect of 3-octanone. G. lucidum was cultivated with 1-octen-3-ol, 3-octanol and 3-octanone for 7 days, after which colony diameter and mycelial dry weight were measured to assess their effects on mycelial growth. RNA-seq was used to investigate gene expression changes following 3-octanone exposure. While 1-octen-3-ol or 3-octanol inhibited mycelial growth in G. lucidum, 3-octanone promoted it. In total, 590 differentially expressed genes (DEGs), including 162 upregulated and 428 downregulated genes, were identified following 3-octanone exposure. Functional annotation revealed that among the DEGs, 23 genes were related to fungal cell wall biosynthesis and remodeling, whereas 21 genes were involved in plant-derived polysaccharide degradation. Furthermore, significant expression changes were observed in genes related to secondary metabolism. Our results indicate that G. lucidum can use 3-octanone as a signal to recognize other fungi, potentially facilitating the expansion of its own territory within wood in nature.

Strains of S. cerevisiae expressing GLP-1-GIP peptide precursors with an N-terminal extension demonstrate up to 29% higher molar productivity during continuous cultivation compared to non-extended counterparts. Additionally, inactivation of the S. cerevisiae AMN1 gene, through deletion, effectively reduces broth viscosity by more than 45%, thereby enhancing oxygen transfer without adversely affecting cell metabolism or product titer. These results highlight the advantages of molecular modifications and genetic alterations for improving the production efficiency of GLP-1 receptor agonists, addressing critical challenges related to recombinant expression, viscosity and oxygen transfer in bioprocessing.

We investigated the effectiveness of hydrated deep eutectic solvents (DESs) to achieve tunable seaweed cellulose microstructures from Ulva lactuca. Cellulose was extracted via a sequential chemical protocol, then treated with 30% DESs (choline chloride (ChCl) or betaine as hydrogen bond acceptors and urea, citric acid, or oxalic acid as donors) combined with mechanical shearing. While most DESs combinations yielded spherical seaweed cellulose microparticles (dry diameter of 605-777 nm), the ChCl:urea formulation successfully produced seaweed cellulose microfibers (SCMFs). These SCMFs exhibited superior quality (dry diameter of 372 nm), and water dispersibility with a hydrodynamic diameter of 134 nm and a polydispersity index of 0.23. Crucially, the DES composition dictated cellulose structure: ChCl:urea-treated SCMF remained predominantly amorphous, whereas other DESs treatments increased microparticles crystallinity. Furthermore, ChCl:oxalic acid introduced carboxyl functional groups. Selecting appropriate hydrated DESs thus offers a sustainable biotechnology tool to tune cellulose morphology, crystallinity, and surface chemistry.

The present study focused on the enhanced biodegradation of phenol as a toxic compound using peroxidases extracted from agricultural wastes (skin peels of luffa and jicama plants) mediated bioprocess under H₂O₂-infusion. The extracted crude luffa peroxidase (LP) and jicama peroxidase (JP) showed enzyme activities of 1.4 ± 0.2 U/mL and 1.6 ± 0.1 U/mL, respectively. LP achieved a phenol removal efficiency exceeding 95% at H₂O₂ concentrations between 6-8 mM and pH 7, whereas JP achieved 94.5% removal at pH 7, with minimal impact from variations in H₂O₂ concentrations. Maximum removal efficiencies for both peroxidases were observed at an enzyme loading of 1.5 mL. LP demonstrated effective removal within a temperature range of 25 °C to 30 °C, while JP maintained functionality up to 40 °C. The optimum reaction times were determined to be 16 h for LP and 13 h for JP. LP exhibited high preservation of enzyme activity during storage, with no significant fluctuations observed, while JP's enzyme activity decreased to approximately 40% of its initial value, accompanied by a notable decline in phenol elimination efficiency by day 42. First-order kinetic analysis of enzyme activity yielded reaction rate constants of 1.18 h^-1 for LP and 1.21 h^-1 for JP, with JP demonstrating superior storage stability compared to LP.

Exopolysaccharide (EPS) is a biopolymer produced by microorganisms. This study aimed to optimize EPS production from lactic acid bacteria (LAB) to enhance the gelation quality of fermented soy milk by varying physical growth conditions. EPS was purified using gel filtration column chromatography, and the proximate composition of the fermented soy milk was analyzed according to AOAC methods. Lactiplantibacillus plantarum JCM 1149 produced a crude EPS yield of 166.4 mg/100 mL under optimized conditions (i.e. 35 °C, pH 4.8, 0.5 mL inoculum) after 24 h, while the purified EPS was 158.6 mg/100 mL with a glucose equivalent of 0.209 g/100 mL. Fermented soy milk produced under these conditions (sample T) was compared to a control prepared with a commercial yogurt starter culture (sample C). Sample T had a higher water-holding capacity (69.1%) and lower syneresis (35.80%) than sample C (30.80% and 41.01%, respectively). No significant differences (p > 0.05) were observed between samples for protein, lipid, nitrogen-free extract, or fiber content. However, sample T showed significantly higher moisture content (80.62 ± 0.30%) and energy (46.64 ± 0.29 kcal) slightly lower ash content (0.37 ± 0.00%) compared to sample C (0.41 ± 0.00%). Total viable counts were similar between samples C (7.10 × 10² cfu/mL) and T (7.60 × 10² cfu/mL). Sensory evaluation indicated that sample T had higher overall acceptability (7.46 ± 0.08) than sample C (6.98 ± 0.10). These results demonstrate that EPS-producing LAB can improve the gelation, functional, and nutritional qualities of fermented soy milk.

Carrot (Daucus carota L.), a key crop in the Apiaceae family, adapts to environmental changes through its endogenous circadian clock, which coordinates physiological processes over a 24-h cycle. As a central physiological activity of carrot growth, photosynthesis, including parameters such as photosynthetic efficiency and stomatal conductance, is influenced by environmental factors. Among them, photoperiod is one of the main regulatory factors for development and chlorophyll synthesis. In this study, we investigated the effects of three different light cycles (16 h of light/8 h of darkness, 16L/8D, 12 h of light/12 h of darkness, 12L/12D, and 8 h of light/16 h of darkness, 8L/16D) on the expression of circadian rhythm genes in carrot. The core clock genes, DcLNK1, DcLNK3, DcRVEa, DcRVEb, and DcGI exhibited distinct transcriptional responses to these light cycles. Three different photoperiod treatments led to changes in the expression profiles of circadian rhythm related genes, photosynthetic parameters, and stomatal activity in carrot. The results elucidated a potential molecular regulatory network linking photoperiod perception to physiological pathway in carrot, providing potential insight into the intrinsic mechanisms of photo-entrainment within its circadian system.

Adeno-associated virus (AAV), a 4.7 kb single-stranded DNA virus, is widely used as a gene therapy vector, but its limited packaging capacity poses challenges for delivering large genes, always resulting in truncation during packaging. Quantifying truncation is difficult because both strands of the plasmid can be packaged from the 3' end. In this article, we aim to first produce single-polarity AAV and then explore its truncation pattern. To address this, we modified one of the ITRs and created an oversized self-complementary AAV, which functions as a single-polarity vector to some extent. Using this modified backbone, we generated a series of oversized single-polarity AAV (spAAV) vectors of varying lengths and sequences, analyzing DNA truncation patterns via quantitative PCR (qPCR). The results show that as the distance from the 3'-ITR increased, less DNA was detected. At 3000 bp from the 3'-ITR, 70% of the genomic DNA remained; this dropped to 50% at 4000 bp, 20% at 4500 bp, and almost none beyond 5000 bp. Additionally, reporter gene expression significantly decreased when the expression cassette extended to 4.5 kb compared to 2.7 kb under identical in vitro conditions. Our results show that DNA will be truncated far earlier before 4.5 kb during the packaging of very large genomes. This study provides important insights into the truncation patterns of AAV genomes, which is crucial for optimizing AAV vector design in gene therapy.

Genistein, a bioactive isoflavone predominantly found in legumes, has garnered considerable attention for its diverse health-promoting benefits. Given the limitations of conventional plant extraction and chemical synthesis, microbial production offers a sustainable and efficient alternative. Endophytic fungi represent a promising reservoir of bioactive metabolites. However, reports on genistein-producing fungal endophytes remain scarce. In this study, the genistein-producing capabilities of endophytes isolated from pigeon pea (Cajanus cajan) were investigated. Five endophytes including three strains of Fusarium solani (G6, G34, G38), F. oxysporum (G44), and Aspergillus sydowii (J11), were identified as novel genistein producers, with A. sydowii J11 being the most effective. Genistein was identified through UV spectroscopy, HPLC, UPLC-MS/MS, and NMR analysis. Under optimized fermentation conditions (potato dextrose broth supplemented with 2% fructose and 1% beef extract, pH 6.0, 28 °C, 10 days), A. sydowii J11 produced genistein at 128.85 mg/L-a 5.4-fold increase over the baseline and the highest microbial genistein yield reported to date. The fungal-derived genistein demonstrated potent antibacterial activity against Staphylococcus aureus and Bacillus subtilis (MIC = 6.25 mg/L), and moderate activity against Escherichia coli and Serratia marcescens (MIC = 12.5 mg/L). Additionally, it exhibited significant antioxidant capacity, with an EC₅₀ of 156.81 mg/L in the DPPH assay. These findings position A. sydowii J11 as a promising candidate for large-scale genistein production and a natural source of antibacterial and antioxidant agents for food and pharmaceutical applications. This work represents the first report of genistein biosynthesis by endophytic fungi from pigeon pea, highlighting the potential of A. sydowii J11 as a sustainable microbial platform for high-yield genistein production.

Phospholipase C enzymes (plcA, plcB, plcC) represent critical virulence determination in mycobacterium tuberculosis pathogenesis. These enzymes play pivotal roles in disrupting phagosomal maturation, inducing macrophage necrosis, and facilitating immune evasion mechanisms. Protein sequences of phospholipase C gene plcA, plcB,plcC were collected and screened against human to exclude homologous matches and minimize cross-reactivity. Linear B-cell epitopes and T cell epitopes were identified and evaluated for the ability to produce strong antigenicity, solubility, toxicity and allergenicity. Suitable segments were linked using EAAK for adjuvant fusion, GPGPG between T-cell epitopes, and AAY between cytotoxin T-cell epitopes, with the L7/L12 ribosomal proteins at N-terminus as immunostimulatory adjuvant, The full vaccine structures were modelled in 3-D and improved for accuracy, and interaction of tuberculosis related proteins were analysed. Immunological potent epitopes for all 3 phospholipase C enzymes with favourable physiochemical properties and structural stability were identified. Immune simulation predicted effective stimulation of both humoral and cell-mediated responses. Molecular docking revealed promising interactions with key targets, characterized by favourable binding energies and stable complex formation dominated by hydrogen bonds and electrostatic interactions, suggesting potential functional efficacy of the designed vaccine. The study presents a strong potential of rationally designed multi-epitope vaccine candidate targeting phospholipase C virulence factor of M. tuberculosis. The computational workflow established a rigorous selection process for immunologically relevant epitopes assembled into chimeric construct with predicted vaccine potential. Further experimental validation through invitro antigenicity assay and in-vivo immunization studies is needed to assess the translational potential of this computationally designed vaccine.

Chitin valorization through microbial bioprocessing relies on efficient utilization of its monomeric units as fermentation substrates. In this study, the effects of salt concentration and the mixing ratio of N-acetylglucosamine (GlcNAc) to glucosamine hydrochloride (GlcN·HCl) on the specific growth rate of our previously isolated V. natriegens N5.3 was investigated in the shake-flask. Batch and fed-batch fermentations using chitin-derived amino sugars were further performed to assess high-cell-density cultivation potential.Although the maximum specific growth rate ( ${\mu }_{\max }$ ) at 60 g/L NaCl was nearly two-fold lower than that at the optimal concentration of 15 g/L, strain N5.3 retained robust growth with ${\mu }_{\max }$ values of 0.37 h^-1 on GlcN·HCl and 0.66 h^-1 on GlcNAc. Fed-batch cultivation yielded a maximum cell dry weight (CDW) of 42.3 g/L within 9 h on GlcNAc, with ${\mu }_{\max }$ of 0.53 h^-1, but with a low biomass yield ( $Y_{X/S}$ = 0.16 g/g). In contrast, a substrate mixture containing 5% (w/w) GlcNAc and 95% (w/w) GlcN·HCl maintained a high ${\mu }_{\max }$ (0.49 h^-1) while substantially improving $Y_{X/S}$ (0.29 g/g), resulting in a CDW of 35.5 g/L after 9 h. Due to low solubility of both amino sugars, exponential feeding with non-sterilized powders was successfully applied. The absence of contamination demonstrate the feasibility of this approach. These results demonstrate that the mixture of GlcNAc:GlcN·HCl (1:19 ratio) is effective substrate for cultivation of V. natriegens N5.3. This provides a promising foundation for the microbial conversion of chitin-derived feedstocks into high-value products.