Biotechnology Letters最新文献

Phage contamination poses a significant threat to industrial fermentation, leading to substantial economic losses. Virulent T-even type phages (T2/T4/T6) represent particularly concerning biological hazards in fermentation systems. This paper developed a novel CRISPR/Cas12a-based system integrated with recombinase polymerase amplification (RPA), enabling ultrasensitive identification of T-even type phages. This method targeted the TerL gene of T-even type phages as a detection marker. The optimized RPA-CRISPR assay demonstrated exceptional sensitivity with a limit of detection (LOD) reaching 1 aM for synthetic targets. Besides, this system achieved detection thresholds of 1 and 10 PFU/μL for T2 and T4 phages, respectively. Comparative validation with quantitative PCR (qPCR) confirmed the method's reliability through strong correlation in the detection for both spiked and wastewater samples. The detection platform exhibited remarkable potential for rapid, sensitive monitoring of T-even type phages contamination in fermentation processes, offering promising application prospects for quality control in biochemical industries.

As critical environmental factors, nitrogen and light not only regulate phytoplankton growth but also influence their phenotypic plasticity. Scenedesmus obliquus, an alga which is famous for its remarkable phenotypic plasticity, was studied to understand its response to varying combinations of nitrogen source and light intensity. It was cultured in media containing different nitrogen sources (NaNO₃, NH₄Cl, CO(NH₂)₂) under a range of light intensities (25, 50, 75, 100, 150 µmol photons m^-2 s^-1). Results showed that growth rates increased with higher light intensities across all nitrogen sources. Photosynthetic efficiency (F_v/F_m and Φ_PSII) remained stable in NaNO₃ treatments, but declined with rising light intensity in NH₄Cl and CO(NH₂)₂ treatments. The highest proportions of multicellular colonies were observed at 150 µmol photons m^-2 s^-1 for NH₄Cl and NaNO₃ treatments, while colonies in CO(NH₂)₂ treatments peaked at 100 µmol photons m^-2 s^-1, with colony size stabilized at approximately 2.1, 4.0, and 1.0 cells per particle under NaNO₃, NH₄Cl, and CO(NH₂)₂ treatments, respectively. Nitrogen removal efficiency improved with increasing light intensity across all treatments, though S. obliquus exhibited varying capacities to remove nitrogen depending on the sources. These findings demonstrated how S. obliquus adapts to varying nitrogen sources and light intensities in its growth, photosynthesis, and morphology, providing new evidence for our insights into its ecological versatility. This study established a theoretical foundation for optimizing culture conditions in applications such as wastewater treatment and bioenergy production.

Vitreoscilla hemoglobin (VHb), a homodimeric bacterial hemoglobin, exhibits distinct oxygen-binding properties that enhance cellular respiration and metabolic activity, particularly under hypoxic conditions. This review presents an updated and comprehensive synthesis of VHb-related research, encompassing its molecular structure, redox biochemistry, and transcriptional regulation. Compared with previous reviews, this work integrates recent mechanistic insights-especially those concerning transcription factor interactions, redox-coupled electron transfer, and structural-function relationships elucidated via targeted mutagenesis. In addition to its canonical role in oxygen delivery, VHb has been increasingly utilized in synthetic biology, high-cell-density fermentation, CRISPR-regulated expression platforms, and mammalian systems. Its biotechnological applications extend to enhancing microbial productivity under oxygen limitation, facilitating biocatalysis, and promoting biodegradation. In addition, the review highlights the emerging application of the vgb promoter as a strong regulatory element and summarizes current trends in VHb-related intellectual property and commercial development. VHb also shows promise in next-generation technologies such as environmental remediation and precision agriculture. Future directions should focus on optimizing expression systems, characterizing protein interaction networks, and engineering modular VHb-based components for advanced biosystems.

Malaria has been a prominent health burden for decades globally. The complex life cycle of Plasmodium made numerous challenges in finding an effective candidate for developing a potent transmission-blocking vaccine (TBV) against malaria. A wide variety of genes of Anopheles mosquitoes' midgut and salivary gland play a pivotal role in the Plasmodium invasion and transmission inside the mosquito body. Targeting mosquitoes' genes offered new insights into developing a more efficient TBV with higher potential to impede the parasite transmission within the Anopheles. Fibrinogen-related protein 1(FREP1) is a mosquito midgut protein that plays a crucial role in parasite transmission. In this study, we opted for an immunoinformatic approach to target An. stephensi FREP1 protein for breaking the parasite cycle so that the life cycle of the parasite could be broken within the mosquito. The FREP1 vaccine was assessed for allergenicity, antigenicity, toxicity, immunogenicity, population coverage, conservancy, solubility, secondary and tertiary structure, which suggested the impeccable quality of the vaccine construct. The interaction between the vaccine and TLR4 receptor via molecular docking revealed an efficient, strong, and stable complex formation. The molecular dynamic simulation and in-silico immunization profiling indicated the remarkable free binding energy and higher potency of the vaccine to generate a significant immune response, respectively. Furthermore, codon optimization and in-silico cloning of the vaccine in Escherichia coli exhibited efficient protein expression. In summary, the FREP1 protein-based multiepitope vaccine can be considered an innovative formulation for targeting the parasite within the vector to impede malaria transmission and vector control as well.

Lipase is a type of hydrolase that catalyzes reactions at the water-in-oil (O/W) interface and possesses significant applied value across various fields. This study introduces integrated reaction-separation system employing microfluidic slug in a water-in-oil (W/O) droplet flow, specifically designed to enhance lipase-catalyzed interfacial lipid hydrolysis. By incorporating spiral microchannels, the system significantly improves interfacial mass transfer through slug flow-induced mixing and turbulence. Tributyrin hydrolysis within a liquid paraffin/phosphate buffer biphasic system serves as the model reaction to investigate the mechanisms underlying the intensification of interfacial enzymatic catalysis. Under comparable conditions, the microfluidic slug droplet system achieves an enzymatic reaction rate approximately 20 times greater than that observed in conventional beaker-based systems and 1.36 times greater than that in straight microchannels. The effects of droplet size, total flow rate, and channel curvature on conversion efficiency and reaction kinetics are examined, demonstrating that these parameters significantly impact mass transfer behavior. The dynamic interfaces generated within the slug flow architecture increase the specific surface area and facilitate accelerated mass transport, thereby enabling more efficient oil-water biphasic catalysis. This platform offers considerable potential for advancing interfacial biocatalysis and optimizing enzymatic transformations across a broad range of industrial and biotechnological applications.

Background: Biofilm formation in Pseudomonas aeruginosa provides protection against multiple stressors and contributes to its pathogenicity. Pyocyanin, a virulence factor regulated by quorum sensing, is crucial for infections. This study aimed to evaluate how various physicochemical conditions impact biofilm formation and pyocyanin production in P. aeruginosa PA14.

Methods: Biofilm formation and pyocyanin production were assessed under varying conditions, including nutrient availability, NaCl concentrations, pH, temperature, heavy metal salts, light exposure, and microbial competition. Biofilm levels were quantified using a crystal violet assay, while pyocyanin levels were measured spectrophotometrically. Statistical analyses were performed to identify significant trends and correlations.

Results: Key findings revealed that biofilm formation and pyocyanin production were reduced under most stress conditions examined in this study, compared to controls, with few exceptions. FeCl₃ enhanced biofilm formation, while NaCl concentrations above 3% and extreme pH values inhibited it. NiCl₂ was the most effective at reducing biofilm amount among the salts which we examined. Pyocyanin production followed similar trends, peaking under neutral pH and nutrient-enriched conditions. Positive correlations between biofilm and pyocyanin production were observed, particularly in nutrient-limited media. Additionally, light exposure and inter-microbial competition significantly reduced biofilm levels.

Conclusion: This study highlights the differential responses of P. aeruginosa to various stress conditions, underscoring the importance of environmental factors in modulating biofilm formation and virulence. These findings provide insights into bacterial adaptive strategies and offer potential avenues for developing targeted interventions against biofilm-associated infections.

Acute myeloid leukemia (AML) is a neoplastic disorder of the myeloid stem cell and is most commonly diagnosed in children and young adults. N-CoR is an essential protein that regulates transcriptional repression in normal myeloid cell development Mutations or loss of function in the N-CoR gene result in the abnormal expression of critical genes involved in cell proliferation, contributing to leukemogenic transformation and the development of malignancy in acute myeloid leukemia subtype M5 (AML-M5). This study was aimed to elucidate the mechanism of N-CoR degradation by O-sialo-glycoprotein endopeptidase (OSGEP), a protease that is active in AML-M5 cells only. The AML-M5-specific proteases were isolated using HPLC size exclusion chromatography and anti-N-CoR OSGEP antibodies. In vitro experiments were performed to test the degradation of recombinant N-CoR protein by OSGEP protease. The protease's identity and composition were analyzed via mass spectrometry. Study involved transfection studies using various cell lines to evaluate the subtype-specific activity of OSGEP based on N-CoR expression levels. Study findings revealed OSGEP protease to cleave N-CoR in AML-M5 cells. Mass spectrometry confirmed the identity and composition of a purified, functionally active form of the OSGEP protease. The transfection studies proved that N-CoR was the only protein of the two that OSGEP protease acted on selectively in AML-M5 cells thus proving its specificity in the subtype of cells. Findings of present study suggests that OSGEP protease-mediated N-CoR degradation is an important factor in the development of AML-M5. Current study highlights N-CoR degradation by OSGEP as a key molecular event in AML-M5 and proposes the N-CoR protease as a potential diagnostic and therapeutic biomarker for this leukemia subtype.

One of the characteristics of actinomycetes, especially streptomycetes, is the high GC content in their genome, which often leads to the failure of heterologous expression in E. coli, and thus hinders in vitro enzyme activity experiments. Therefore, we have developed a precisely regulated and efficient Streptomyces expression system, pTZYp, that relied on the strong promoter stnY and the cmt operon. As tested in three model Streptomyces strains (S. albus J1074, S. coelicolor M1152 and S. lividans TK24), the reporter protein sfGFP was not detected without the addition of the inducer cumate, whereas sfGFP was significantly produced when a certain amount of inducer cumate was added to the medium, demonstrating that the pTZYp expression system can achieve the goal of precise regulation and efficient expression. After optimization of the expression conditions, the maximum sfGFP production was obtained when the inducer was added to the final concentration of 100 μM and cultivated for about 24 h. pTZYp has also been used to express other six non-model proteins in Streptomyces, and all of them have been successfully expressed. The pTZYp expression system demonstrated robustness, high efficiency (relying on the stnY promotor), precise regulation (relying on cmt operon and moderate production of regulatory protein CymR) and low experimental cost (relying on the lower cost of the inducer cumate), which may be an efficient and widely applicable heterologous expression tool for genes with high GC content in actinomycetes.

Paramecium caudatum is a well-known ecotoxicological indicator for monitoring heavy metal pollution, including lead contamination. This study investigates P. caudatum's stress response to lead nitrate through the accumulation of polyhydroxyalkanoates (PHAs), biopolymers with potential applications in sustainable bioplastic production. The effect of lead nitrate stress (23.1 g/L) on PHA synthesis was compared with glucose supplementation (180 g/L) and unsupplemented controls. PHA was extracted at 24, 48, and 72 h from both glucose- and metal-treated cultures. Intracellular PHA granules were visualized using Sudan Black B and Nile Blue A staining. Extracted polymers were characterized by FTIR and GC-MS, confirming their chemical identity and structural similarity to poly(3-hydroxybutyrate) (PHB). Scanning electron microscopy (SEM) was used to examine the polymer microstructure. Lead nitrate treatment induced the highest PHA yield (4.8 g/L) after 24 h, significantly exceeding glucose-supplemented (3.8 g/L) and control (1.8 g/L) cultures. FTIR spectra revealed characteristic O-H, C-H, and ester carbonyl (C = O) absorption bands typical of PHB, while GC-MS identified 3-hydroxybutyrate as the dominant monomer along with minor medium-chain-length components. SEM imaging showed a porous, pseudospherical, and interconnected polymer morphology. P. caudatum responds to heavy metal stress by enhancing PHA accumulation, producing high-quality biopolymers structurally comparable to PHB. These findings highlight the dual potential of P. caudatum for bioplastic production and environmental remediation, offering a novel green biotechnology approach to valorize pollutant stress responses.

Lignin peroxidase (LiP) is a key enzyme involved in lignin degradation. However, the production of natural LiP is limited by the low enzyme-producing capacity of native fungal hosts and the poor stability of the enzyme, hindering its industrialization. To overcome these limitations, we prepared an immobilized enzyme by fusing the LiP gene with the Bacillus subtilis (B. subtilis) spore coat protein CotB and displaying it on the bacterial surface. This approach aims to improve enzymatic stability and industrial applicability. Enzymatic characterization showed that the immobilized LiP exhibited optimal activity at 50 °C and pH 5.0, representing a 10 °C increase over the optimal temperature (40 °C) and a lower pH optimum compared to free LiP (pH 6.0). Under extreme conditions (70 °C, 6 h), the free enzyme retained less than 10% residual activity, whereas CotB-LiP maintained 42.7% ± 1.37% activity. After 1 h incubation in 1 mM H₂O₂, CotB-LiP retained 60% relative activity, while free LiP was almost completely inactivated. These findings indicate that spore surface-displayed LiP has significant application potential in environmental remediation and agriculture and provides a reference strategy for low-cost immobilized enzyme production.