Biotechnology Letters最新文献

The reduction of quinones and nitroaromatic compounds catalyzed by Type I nitroreductases is important due to its role in their potential cytotoxic effects and/or biodegradation. The main goal of this work was to investigate the mechanism of catalysis of a TdsD nitroreductase (NR) (TdsD1), a member from an understudied branch of the nitroreductase superfamily, derived from a soil metagenome study. Like the Type I NRs NfsA and NfsB, TdsD1 performed two-electron reduction of quinones and four-electron reduction of nitroaromatic compounds according to a "ping-pong" mechanism with a rate-limiting oxidative half-reaction. TdsD1 was also inhibited by the classical inhibitors of other NRs, dicoumarol and Cibacron blue. Despite sharing only a low degree of homology with the NfsA and NfsB subfamily enzymes, sequence comparisons and computer modelling point to the possibility of an analogous FMN isoalloxazine ring location within the intersubunit space of TdsD1. It also possesses similar specificity for nitroaromatic compounds and quinones, in particular the shared characteristic of being especially active with 2-hydroxy-1,4-naphthoquinone derivatives. It is possible that the similar character of binding of oxidants and other ligands relative to the NfsA and NfsB subfamily enzymes may be related to the conserved Arg27 and Ser53 residues in the active site of TdsD1.

Currently, Latin America is experiencing a considerable increase in demand for veterinary products to ensure animal health. In particular, interferons (IFNs) are cytokines that play an important role in small animal clinics for the treatment of viral or oncological diseases. We developed a biotechnological process for the production of recombinant canine interferon CaIFN-α7 (rCaIFN-α7) using the baculovirus-insect cell and larvae system, and evaluated its biological activity, both antiviral and antitumor, for comparison with the veterinary interferon available on the market, feline omega interferon (rFeIFN-ω, Virbagen Omega®). Recombinant rCaIFN-α7 expressed in Sf9 cell culture was purified in a single step by immobilized metal ion chromatography, yielding 4.43 mg/l with 95% purity. The purified rCaIFN-α7 showed antiviral activity against the canine kidney cell line MDCK (NBL-2, Madin Darby canine kidney) derived from Canis lupus familiaris kidney (ATCC NBL-2) infected with Vesicular Stomatitis Virus (VSV, New Jersey strain), with a recovery of 5.34 × 10⁷ AU/ml after purification. Furthermore, rCaIFN-α7 exhibited antitumor activity against canine mucosal melanoma cell lines Ak and Bk. In a typical scale-up process using 200 Rachiplusia nu larvae, biologically active rCaIFN-α7 was obtained, showing an antiviral titer of 4.5 × 10⁷ AU/ml and a yield of 5.36 × 10⁶ AU/g of larvae. This product also showed antitumor activity against canine mucosal melanoma cell line Mc and a high degree of purity from the starting sample, but a yield of 20%, probably due to aggregation or degradation events leading to a decrease in biological activity.

Effective mosquito control is essential for reducing the transmission of vector-borne diseases. This study focuses on the comprehensive characterization of mosquitocidal toxins produced by Bacillus thuringiensis serovar israelensis (Bti) VCRC B646 and the associated insecticidal genes. The bacterium was cultured, and the spore-crystal complex was purified to identify the mosquitocidal proteins. The mosquitocidal toxins produced by the isolate were evaluated against Aedes(Ae.) aegypti and Culex(Cx.) quinquefasciatus. SDS-PAGE and LC-MS were conducted to analyse the toxin, and PCR was performed to amplify the toxin gene. Toxicity bioassays indicated lethal concentrations (LC₅₀ and LC₉₀) for Ae. aegypti (0.0022 mg/L and 0.004 mg/L), and Cx, quinquefasciatus (0.0025 mg/L and 0.0044 mg/L). SDS-PAGE and LC-MS analysis revealed that Cry11Aa5 (Pesticidal Crystal Protein) is the predominant toxin produced by this strain. PCR amplification confirmed the presence of genes encoding various insecticidal proteins, including Cry and Cyt toxins. Phylogenetic analysis was performed to assess the genetic relatedness and toxin profiles of the bacterial isolate. This detailed characterization of Bti VCRC B646 highlights its potential as a promising biopesticide candidate for mosquito control, contributing to the development of sustainable and eco-friendly strategies for vector management.

The cell surface display system employs carrier proteins to present target proteins on the outer membrane of cells. This system enables functional proteins to be exposed on the exterior of living cells without cell lysis, allowing direct interaction with the surrounding environment. A major limitation of conventional approaches is the difficulty in displaying large-sized enzymes or antibodies, despite their critical roles in applications requiring functional domains that must remain intact, such as catalytic or antigen-binding sites. To address this challenge, we developed a novel system that enables the surface presentation of target proteins in Escherichia coli by integrating the cell surface display system with the self-assembly of split green fluorescent proteins (GFPs). In this system, GFP11M3 was fused to the carrier protein Lpp-OmpA and displayed on the bacterial surface. The surface-localized Lpp-OmpA-GFP11M3 subsequently assembled with GFP1-10opt, forming a functional GFP complex. By conjugating other target proteins, such as enzymes or antibodies, to GFP1-10opt, these biomolecules can be efficiently displayed on the cell surface. This approach not only facilitates the presentation of large biomolecules but also enables real-time visualization of protein localization through fluorescence detection.

Plasmalogens are a subclass of glycerophospholipids characterized by a vinyl-ether bond at the sn-1 position; they play several physiological roles including membrane stabilization, antioxidant activity, and signal transduction. While choline, ethanolamine, serine, and glycerol plasmalogens (PlsCho, PlsEtn, PlsSer, and PlsGro) are naturally abundant, inositol plasmalogens (PlsIns) are rare. In contrast to the limited occurrence of PlsIns, phosphatidylinositol is a biologically crucial lipid, and its enzymatic biosynthesis from phosphatidylcholine has been extensively studied. Given that inositol itself is known to exert a range of physiological effects, it is reasonable to expect that PlsIns may also possess distinctive biological functions. Here, we report the first enzymatic synthesis of PlsIns using a phospholipase D-mediated transphosphatidylation reaction. Plasmalogen substrates-PlsEtn from Selenomonas ruminantium and both PlsEtn and PlsCho from chicken breast-were successfully converted to novel PlsIns species in the presence of myo-inositol. The resulting plasmalogens were detected by liquid chromatography-tandem mass spectrometry, confirming the introduction of the inositol moiety into the head group region. The results indicated that our method can be applied to different types of plasmalogens with different head groups and fatty acid profiles, including chain length and degree of unsaturation. This finding opens new avenues for exploring PlsIns and their potential biosignificance.

Fatty acid synthase (FAS) is one of the most important enzymes in lipid biosynthesis, which can catalyze the reaction of acetyl-CoA and malonyl-CoA to produce fatty acids. However, the structure, function, and molecular mechanism of FAS regulating lipid synthesis in the fungus Mucor circinelloides are unclear. In the present study, two encoding fas genes in the high lipid-producing strain WJ11 and low lipid-producing strain CBS277.49 from M. circinelloides based on their genome database were identified and annotated respectively. Bioinformatic analysis confirmed the presence of typical conserved domains in FAS proteins. Phylogenetic analysis revealed the evolutionary relationship of these FAS proteins, and the FAS proteins from WJ11 have similar properties as their counterparts in CBS277.49. Furthermore, transcriptional profiling displayed marked differences in the expression of these fas genes, and fas1 was analyzed to predict the possible functions in lipid metabolism in the high lipid-producing strain WJ11. This is the first report on the structures and functions of FAS proteins in M. circinelloides. This research has suggested the association of fas genes with lipid metabolism at the transcriptional level and contributed to the selection of candidates related to lipid metabolism for further study.

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.