Biotechnology Letters最新文献

Objective: Adeno-associated viruses (AAVs) are widely used as gene therapy vectors due to their safety, stability, and long-term expression characteristics. The objective of this work is to develop an aqueous two-phase system (ATPS) as a universal platform for the separation and purification of AAVs.

Results: This study utilized polyethylene glycol (PEG)/salt ATPSs to separate and purify various AAV serotypes, including AAV5, AAV8, and AAV9, which focusing on serotype-specific performance and partial empty capsid removal. The results showed that all the AAV serotypes were mainly enriched in the interphase of ATPS, with achieving high recovery (> 95%) and impurity removal (> 95%). The PEG/sodium citrate ATPS was serotype-independent, but the process optimization of component concentrations for each serotype was necessary to attain the best performance. Notably, a single-step aqueous two-phase extraction also demonstrated the ability to remove some amount of empty capsids from the crude cell lysate, with removal rate ranging from 4 to 25%.

Conclusions: The results demonstrated the practical applicability of PEG/sodium citrate ATPS in separating and purifying different AAV serotypes, which addressing key challenges in gene therapy vector production.

Recombineering (recombination-mediated genetic engineering) is a powerful strategy for bacterial genomic DNA and plasmid DNA modifications. CoS-MAGE improved over MAGE (multiplex automated genome engineering) by co-electroporation of an antibiotic resistance repair oligo along with the oligos for modification of the Escherichia coli chromosome. After several cycles of recombineering, the sub-population of mutants were selected among the antibiotic resistant colonies. However, a pre-generated strain with mutS deletion and multiple inactivated antibiotic resistance genes integration is required. Herein, CoS-MAGE was modified by employing a single copy BAC vector harboring a bla-mkan cassette and a Red helper vector cloned with dominant mutL E32K, thus bypassing the utilization of the pre-generated strain. The proof-of-concept of the new strategy, CoS-BAC-MAGE, was demonstrated via the mutation of non-essential genes, essential genes, and AT rich regions of the wild type strain E. coli MG1655. With this system, an editing efficiency of 60% was realized. Furthermore, by toggling between two antibiotic resistance genes (one active, the other defective) on the BAC, sequential mutations were achieved without the requirement of BAC vector elimination and re-transformation. Via CoS-BAC-MAGE, simultaneously mutations of three sites were obtained in a day. We envision that CoS-BAC-MAGE will be a practical improvement for the generation of chromosomal mutations using the Cos-MAGE approach.

Rapid diagnostic tools for Porphyromonas gingivalis (Pg), the primary microorganism responsible for the development of periodontitis, particularly those designed for chair-side applications, could provide substantial health benefits to patients. To address this issue, we developed a CRISPR/Cas12a-based rapid Pg detection method. Dual-gRNA and hairpin reporter strategies were employed to enhance CRISPR/Cas12a reaction efficiency. By modifying the hairpin reporter with HRP, the pre-amplification-free HRP-CRISPR/Cas12a reaction was enabled to produce a colorimetric output, amplifying the detection signal. This method achieved high sensitivity (as low as 33 CFU) without the risk of aerosol contamination from pre-amplification. When testing clinical samples, the method showed high consistency with the reference RT-PCR. Furthermore, compared with RT-PCR, this method only requires room temperature operation, is simpler, and has a shorter detection time of about 35 min. In conclusion, the pre-amplification-free HRP-integrated CRISPR/Cas12a detection method requires no complex equipment, making it an ideal, end-user-friendly approach for chair-side Pg detection.

Objectives: Pseudomonas aeruginosa, identified as an ESKAPE pathogen, contributes to severe clinical diseases worldwide and despite its prevalence an effective vaccine or treatment remains elusive. Numerous computational methods are being employed to target hypothetical proteins (HPs). Presently, no studies have predicted multi-epitope vaccines for these HPs.

Results: Totally, 877 HPs from P. aeruginosa were included in the study and the data showcased here illustrate a methodical approach to prioritize the proteome by employing diverse comparative proteomics. The study employed physicochemical property assessment and conserved domain analysis to identify stable and immunologically pertinent proteins for epitope prediction. The VaxiJen2.0 antigenicity assessment aided in epitope selection, contributing to the foundational steps in vaccine development by predicting T-cell and B-cell epitopes. Potential T and B cell epitopes with high antigenicity, non-toxic categorization, and robust binding affinities were identified in the investigation. The periplasmic HP WP_132813935.1 was predicted as conserved, stable, and soluble. The T-cell peptide RTSMRALAY and the B-cell peptide MPVYLYLM were predicted to be probable non-allergen and demonstrated strong binding with MHC class I allele HLA-C*03:03.

Conclusions: This research provides a comprehensive approach to predict T and B cell epitopes for conditions associated with P. aeruginosa, offering a candidate pool for tailored vaccine development. However, the efficacy of these epitopes in vaccine development necessitates clinical validation and testing for confirmation.

In this study, the crude chemical constituents extracted from Trichoderma harzianum and their toxicity were evaluated against the larvae, pupae, and adults of Anopheles stephensi at 24 and 48 h post-treatment. Additionally, the chemical constituents of the crude extracts were identified using gas chromatography-mass spectrometry (GC-MS) analysis, and their ability to bind with target proteins was confirmed through molecular docking studies. The results clearly demonstrated that the chemical compounds from T. harzianum exhibited promising mortality rates in larvae (98.66%), pupae (92%), and adult mosquitoes (81.33%) of A. stephensi 48 h after treatment. The study assessed the impact of crude extracts on insect enzymes 24 h post treatment, revealing significant alterations: a reduction in catalase activity and an increase in glutathione S-transferase levels compared to the control group. The treatment with crude chemical extracts resulted in mortality rates of 37.33% and 52% at 24 and 48 h, respectively, on Artemia salina , indicating minimal effects. After 48 h, the crude extract exhibited minimal toxicity on Eudrilus eugeniae, with a recorded mortality rate of 15% after 48 h. GC-MS analysis of T. harzianum-derived crude extracts identified ten major chemical constituents. Among these, chemicals, 2,4-bis(1,1-dimethylethyl) phenol (19.02%) was recognized as the predominant chemical component. This 2,4-bis(1,1-dimethylethyl) phenol molecule demonstrates a high binding affinity with target proteins, which is a key factor contributing to its insecticidal activity. This study concludes that the chemical constituents derived from T. harzianum are promising candidates for an eco-friendly, effective, and target-specific alternative control method for A. stephensi mosquitos.

Objectives: To develop robust variants of L-threonine aldolases (L-TAs), potent catalysts for synthesizing asymmetric β-hydroxy-α-amino acids, it is necessary to identify critical residues beyond the known active site residues.

Results: Through virtual screening, a neglected residue Asn305, was identified as critical for catalytic efficiency. Subsequent site-saturation mutagenesis led to a potent variant N305R which exhibited excellent conversions of 88%_conv (87%_de) and 80%_conv (94%_de) for the synthesis of L-threo-phenylserine and L-threo-4-fluorophenylserine respectively. This variant not only outperformed the template enzyme, but also represented a promising L-TA for synthesizing the two β-hydroxy-α-amino acids. It was suggested that Arg305 of the variant N305R generated strong cation-arene interaction and electrostatic force with the intermediates, leading to strengthened binding, enhanced L-threo favored orientation and wider entrance.

Conclusions: Our work not only provided an excellent variant N305R, but also suggested the crucial function of a neglected residue Asn305, which offered valuable experiences for other L-TA studies.

The cell walls of red and green algae contain β-1,3-xylan, which is hydrolysed by the endo-type enzyme β-1,3-xylanase. Notably, only marine-bacteria-derived β-1,3-xylanases have been functionally characterised to date. In this study, we characterised the enzymatic properties of a potential β-1,3-xylanase (BcXyn26B) derived from the human gut bacterium, Bacteroides cellulosilyticus WH2. The codon optimized BcXyn26B gene was synthesised and expressed in Escherichia coli BL21(DE3). The recombinant protein was purified by a two-step purification process using Ni-affinity chromatography followed by anion exchange chromatography, and its enzymatic properties were characterised. The recombinant BcXyn26B exhibited specific hydrolytic activity against β-1,3-xylan and released various β-1,3-xylooligosaccharides, with β-1,3-xylobiose as the primary product. The optimum reaction temperature was 50 °C, higher than that for other enzymes derived from marine bacteria. This study represents the first report on the identification, heterologous expression, and characterisation of β-1,3-xylanase from human gut microbes. Notably, the substrate specificity of BcXyn26B indicates that human gut Bacteroides species possess an unknown β-1,3-xylan utilisation system.

The α-L-rhamnosidase (rha1) gene was homologously expressed in Aspergillus niger strains CCTCC 206047 and CCTCC 206047ΔpyrG, using hygromycin B and auxotrophic as selection markers. The engineered A. niger strains RHA001-1 and RHA003-1 were screened, yielding α-L-rhamnosidase activities of 20.81 ± 0.56 U/mL and 15.35 ± 0.87 U/mL, respectively. The copy numbers of the rha1 gene in strains RHA001-1 and RHA003-1 were found to be 18 and 14, respectively. Correlation analysis between copy number and enzyme activity in the A. niger strains revealed that α-L-rhamnosidase activity increased with the copy number of the rha1 gene. Recombinant α-L-rhamnosidase was utilized for the enzymatic debittering of Ougan juice, and its process conditions were optimized. Furthermore, the primary bitter substance neohesperidin (2.22 g/L) in Ougan juice was converted into hesperetin 7-O-glucoside (1.47 g/L) and hesperidin (0.143 g/L). This study presents a novel approach for the production of food-grade α-L-rhamnosidase and establishes a technical foundation for its application in the beverage industry.

The current knowledge about Palestinian safflower landraces is relatively limited in terms of phenotypic and molecular characterization, however, the purpose of this investigation was to determine the amount of genetic diversity in eighteen local safflower landraces using seven DAMD markers. The banding patterns for each primer were scored and compiled into a data matrix. Subsequently, the data matrix was analyzed using UPGMA cluster analysis to identify distinct genetic groups among the landraces. In total, 88 DNA fragments were found, and there were an average of 12.6 loci per assay unit observed. Resolving Power (RP) revealed an average of 7.09 was determined, with the highest RP value at 13.3. The dendrogram obtained from DAMD data divided the landraces into three main clusters, denoted as I, II and III. The first cluster (I) consisted of one genotype (PTUK.SA16). The second cluster (II) consisted of two genotypes (PTUK.SA13 and PTUK.SA10). The third cluster (III) was later partitioned into two distinct sub-clusters, which are III.a and III.b. Sub-cluster III.a comprised seven genotypes (PTUK.SA4, PTUK.SA9, PTUK.SA8, PTUK.SA7, PTUK.SA6, PTUK.SA5 and PTUK.SA3). While Sub-cluster III.b consisted of eight genotypes (PTUK.SA15, PTUK.SA18, PTUK.SA17, PTUK.SA14, PTUK.SA12, PTUK.SA2, PTUK.SA11, and PTUK.SA1). This research assess the genetic diversity of Palestinian safflower landraces using PCR-based DAMD markers. The remarkable level of polymorphism detected using DAMD markers demonstrated their effectiveness in distinguishing between Palestinian safflower genotypes.

The cancer is one of the diseases of serious threat to people's health and life nowadays. But heterogeneity, drug resistance and treatment side effects of cancer, traditional treatments still have limitations. Tumor-targeting probiotics with a well-established Biosafety and efficient targeting as a delivery vectors to deliver anticancer genes or antitumor drugs to tumor microenvironment has attracted much attention in cancer therapies. In this study, E.coil Nissle 1917 (EcN) was utilized to deliver eukaryotic anti-tumor protein PTEN to tumor microenvironment and suppress tumor growth. Therefore, the EcN (PTEN) was developed. Our results demonstrated that EcN (PTEN) could colonize the tumor site accurately and inhibit the growth of colorectal cancer cells in tumor-bearing mice. It is worth noting that the tumor microenvironment of the treated mice showed significant recruitment of and M1 macrophages, neutrophils and T lymphocytes. No toxicity was observed in the normal tissues during the experiments. This research show the probiotic EcN(PTEN) holds the promise of becoming a powerful weapon against cancer and expected to provide more effective treatments for cancer patients.