Journal, genetic engineering & biotechnology最新文献

Background: Human insulin was the first FDA-approved biopharmaceutical drug produced through recombinant DNA technology. The previous studies successfully expressed recombinant human insulin precursors (HIP) in Pichia pastoris truncated and full-length α-factor recombinant clones. The matting α-factor (Matα), a signal secretion, direct the HIP protein into the culture media. This study aimed to compare the HIP expression from full-length and truncated α-factor secretory signals clones that grown in two types of media, buffered methanol complex medium (BMMY) and methanol basal salt medium (BSMM).

Results: ImageJ analysis of the HIP's SDS-PAGE shows that the average HIP expression level of the recombinant P. pastoris truncated α-factor clone (CL4) was significantly higher compared to the full-length (HF7) when expressed in both media. Western blot analysis showed that the expressed protein was the HIP. The α-factor protein structure was predicted using the AlphaFold and visualized using UCSF ChimeraX to confirm the secretion ability for both clones.

Conclusions: CL4 clone, which utilized a truncated α-factor in the P. pastoris HIP expression cassette, significantly expressed HIP 8.97 times (in BMMY) and 1.17 times (in BSMM) higher than HF7 clone, which used a full-length α-factor secretory signal. This research confirmed that deletion of some regions of the secretory signal sequence significantly improved the efficiency of HIP protein expression in P. pastoris.

Background: Over the years, excessive use of chemical pesticides to control plant pathogens has caused environmental problems. Therefore, biological solutions such as the use of microorganisms with antimicrobial capacity become indispensable. To inhibit the growth of plant pathogens, biological control agents use different mechanisms, including the production of hydrolytic enzymes. In this study, the production of amylase, an enzyme important for the prevention and control of plant diseases, by a biological control agent Bacillus halotolerans RFP74 was optimized using response surface methodology.

Results: Bacillus halotolerans RFP74 inhibited the growth of various phytopathogens including Alternaria and Bipolaris with an inhibition rate of more than 60%. In addition, it also demonstrated an essential production of amylase. Based on previous studies of amylase production in Bacillus, three parameters were considered significant: initial pH of the medium, incubation time, and temperature. Using the central composite design with Design Expert software, the optimized amylase production for B. halotolerans RFP74 is at a temperature of 37 °C, incubation time 51 h and pH 6.

Conclusion: The biological control agent B. halotolerans RFP74 inhibited the growth of Alternaria and Bipolaris, demonstrating its broad spectrum of activity. Knowledge of the optimal condition required for the production of hydrolytic enzymes such as amylase provides information on the most effective application of this biological control agent.

Background: Arsenic, a ubiquitous toxic metalloid, is a threat to the survival of all living organisms. Bioaccumulation of arsenic interferes with the normal physiological pathway. To overcome arsenic toxicity, organisms have developed arsenite methyltransferase enzyme, which methylates inorganic arsenite to organic arsenic MMA (III) in the presence of S-adenosylmethionine (SAM). Bacteria-derived arsM might be horizontally transported to different domains of life as arsM or as3mt (animal ortholog). A systematic study on the functional diversity of arsenite methyltransferase from various sources will be used in arsenic bioremediation.

Results: Several arsenite methyltransferase protein sequences of bacteria, fungi, fishes, birds, and mammals were retrieved from the UniProt database. In silico physicochemical studies confirmed the acidic, hydrophilic, and thermostable nature of these enzymes. Interkingdom relationships were revealed by performing phylogenetic analysis. Homology modeling was performed by SWISS-MODEL, and that was validated through SAVES-v.6.0. QMEAN values ranged from - 0.93 to - 1.30, ERRAT score (83-96), PROCHECK (88-92%), and other parameters suggested models are statistically significant. MOTIF and PrankWeb discovered several functional motifs and active pockets within the proteins respectively. The STRING database showed protein-protein interaction networks.

Conclusion: All of our in silico studies confirmed the fact that arsenite methyltransferase is a cytosolic stable enzyme with conserved sequences over a wide range of organisms. Thus, because of its stable and ubiquitous nature, arsenite methyltransferase could be employed in arsenic bioremediation.

Background: Calf rennet is considered the traditional source of milk clotting enzyme (MCE). However, increasing cheese consumption with decreasing the calf rennet supply had encouraged the quest for new rennet alternatives. The purpose of this study is to acquire more information about the catalytic and kinetic properties of partially purified Bacillus subtilis MK775302 MCE and to assess the role of enzyme in cheese manufacture.

Results: B. subtilis MK775302 MCE was partially purified by 50% acetone precipitation with 5.6-fold purification. The optimum temperature and pH of the partially purified MCE were 70 °C and 5.0, respectively. The activation energy was calculated as 47.7 kJ/mol. The calculated Km and Vmax values were 36 mg/ml and 833 U/ml, respectively. The enzyme retained full activity at NaCl concentration of 2%. Compared to the commercial calf rennet, the ultra-filtrated white soft cheese produced from the partially purified B. subtilis MK775302 MCE exhibited higher total acidity, higher volatile fatty acids, and improved sensorial properties.

Conclusions: The partially purified MCE obtained in this study is a promising milk coagulant that can replace calf rennet at a commercial scale to produce better-quality cheese with improved texture and flavor.

Adeno-associated virus (AAV) vectors have emerged as the leading delivery platforms for gene therapy. Throughout the life cycle of the virions, the capsid vector carries out diverse functions, ranging from cell surface receptor engagement, cellular entry, endosomal escape, nuclear import to new particle packaging, and assembly. Each of these steps is mediated by exquisite structure features of the viral capsid and its interaction with viral genome, Rep proteins, and cellular organelle and apparatus. In this brief review, we provide an overview of results from over a decade of extensive biophysical studies of the capsid employing various techniques. The remaining unaddressed questions and perspective are also discussed. The detailed understanding of the structure and function interplay would provide insight to the strategy for improving the efficacy and safety of the viral vectors.

CRISPR-Cas9 is a popular gene-editing tool that allows researchers to introduce double-strand breaks to edit parts of the genome. CRISPR-Cas9 system is used more than other gene-editing tools because it is simple and easy to customize. However, Cas9 may produce unintended double-strand breaks in DNA, leading to off-target effects. There have been many improvements in the CRISPR-Cas system to control the off-target effect and improve the efficiency. The presence of a nuclease-deficient CRISPR-Cas system in several bacterial Tn7-like transposons inspires researchers to repurpose to direct the insertion of Tn7-like transposons instead of cleaving the target DNA, which will eventually limit the risk of off-target effects. Two transposon-encoded CRISPR-Cas systems have been experimentally confirmed. The first system, found in Tn7 like-transposon (Tn6677), is associated with the variant type I-F CRISPR-Cas system. The second one, found in Tn7 like-transposon (Tn5053), is related to the variant type V-K CRISPR-Cas system. This review describes the molecular and structural mechanisms of DNA targeting by the transposon-encoded type I-F CRISPR-Cas system, from assembly around the CRISPR-RNA (crRNA) to the initiation of transposition.

Background: Sudangrass (Sorghum sudanense) is a major biomass producer for livestock feed and biofuel in many countries. It has a wide range of adaptations for growing on marginal lands under biotic and abiotic stresses. The immature inflorescence is an explant with high embryogenic competence and is frequently used to regenerate different sorghum cultivars. Caffeic acid O-methyl transferase (COMT) is a key enzyme in the lignin biosynthesis pathway, which limits ruminant digestion of forage cell walls and is a crucial barrier in the conversion of plant biomass to bioethanol. Genome editing by CRISPR/Cas9-mediated mutagenesis without a transgenic footprint will accelerate the improvement and facilitate regulatory approval and commercialization of biotech crops.

Methods and results: We report the overcome of the recalcitrance in sudangrass transformation and regeneration in order to use genome editing technique. Hence, an efficient regeneration system has been established to induce somatic embryogenesis from the immature inflorescence of two sudangrass cultivars on four MS-based media supplemented with different components. Our results indicate an interaction between genotype and medium composition. The combination of Giza-1 cultivar and M4 medium produces the maximum frequency of embryogenic calli of 80% and subsequent regeneration efficiency of 22.6%. Precise mutagenesis of the COMT gene is executed using the CRISPR/Cas9 system with the potential to reduce lignin content and enhance forage and biomass quality in sudangrass.

Conclusion: A reliable regeneration and transformation system has been established for sudangrass using immature inflorescence, and the CRISPR/Cas9 system has demonstrated a promising technology for genome editing. The outcomes of this research will pave the road for further improvement of various sorghum genotypes to meet the global demand for food, feed, and biofuels, achieving sustainable development goals (SDGs).

Background: Epigenome, genetic variants, and other environmental factors involved in gene regulation are highly inter-dependent in several chronic diseases, including obesity, cardiovascular disease, and diabetes. The present study aimed at testing the associations and the mechanism involved in silencing of CYP2R1 gene in normal and obese Saudi women patients. Height, weight, BMI, 25-hydroxy vitamin D, parathyroid hormone, glycemic status, and lipid profile (TG, LDL, HDL, and TC) of CYP2R1 were measured in 100 women (31 normal and 69 obese patients).

Results: Our result shows that hypermethylation in site 2 of the CYP2R1 gene with body weight (p < 0.004), BMI (p < 0.002), waist circumference (p < 0.002), total-LDL (p < 0.027), total cholesterol (p < 0.022), and vitamin D (VD) (close to borderline significance p < 0.06) and site 4 of CYP2R1 with LDL (p < 0.041) in the four tested sites among normal and obese women was significantly associated. Moreover, we tested five different CpG sites in the CYP27B1 gene where site 5 correlated significantly with VD levels.

Conclusion: Our present study clearly indicates that hypermethylation of specific sites in the CYP2R1 and CYP27B1 genes might regulate gene expression with special reference to the risk of obesity and vitamin D metabolism.

Background: Streptococcus pneumoniae (SPN) is the agent responsible for causing respiratory diseases, including pneumonia, which causes severe health hazards and child deaths globally. Antibiotics are used to treat SPN as a first-line treatment, but nowadays, SPN is showing resistance to several antibiotics. A vaccine can overcome this global problem by preventing this deadly pathogen. The conventional methods of wet-laboratory vaccine design and development are an intense, lengthy, and costly procedure. In contrast, epitope-based in silico vaccine designing can save time, money, and energy. In this study, pneumococcal surface protein A (PspA), one of the major virulence factors of SPN, is used to design a multi-epitope vaccine.

Methods: For designing the vaccine, the sequence of PspA was retrieved, and then, phylogenetic analysis was performed. Several CTL epitopes, HTL epitopes, and LBL epitopes of PspA were all predicted by using several bioinformatics tools. After checking the antigenicity, allergenicity, and toxicity scores, the best epitopes were selected for the vaccine construction, and then, physicochemical and immunological properties were analyzed. Subsequently, vaccine 3D structure prediction, refinement, and validation were performed. Molecular docking, molecular dynamic simulation, and immune simulation were performed to ensure the binding between HLA and TLR4. Finally, codon adaptation and in silico cloning were performed to transfer into a suitable vector.

Results: The constructed multi-epitope vaccine showed a strong binding affinity with the receptor molecule TLR4. Analysis of molecular dynamic simulation, C-immune simulation, codon adaptation, and in silico cloning validated that our designed vaccine is a suitable candidate against SPN.

Conclusion: The in silico analysis has proven the vaccine as an alternative medication to combat against S. pneumoniae. The designated vaccine can be further tested in the wet lab, and a novel vaccine can be developed.

Background: Garlic (Allium sativum L.) is the second most widely cultivated Allium which is mainly grown in temperate regions and used as a flavoring agent in a wide variety of foods. Garlic contains various bioactive compounds whose metabolic pathways, plant-pathogen interactions, defensive genes, identify interaction networks, and functional genomics were not previously predicted in the garlic at the genomic level. To address this issue, we constructed two garlic Illumina 2000 libraries from tissues of garlic clove and leaf.

Results: Approximately 43 million 125 bp paired-end reads were obtained in the two libraries. A total of 239,973 contigs were generated by de novo assembly of both samples and were compared with the sequences in the NCBI non-redundant protein database (Nr). In total, 42% of contigs were matched to known proteins in public databases including Nr, Gene Ontology (GO), and Cluster Orthologous Gene Database (COG), and then, contigs were mapped to 138 via functional annotation against the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). In addition, a number of regulatory genes including the CCHC (Zn) family, followed by WD40, bromodomain, bZIP, AP2-EREBP, BED-type (Zn) proteins, and defense response proteins related to different conserved domains, such as RGA3, NBS-LRR, TIR-NBS-LRR, LRR, NBS-ARC, and CC-NBS-LRR were discovered based on the transcriptome dataset. We compared the ortholog gene family of the A. sativum transcriptome to A. thaliana, O. sativa, and Z. mays and found that 12,077 orthologous gene families are specific to A. sativum L. Furthermore, we identified genes involved in plant defense mechanisms, their protein-protein interaction network, and plant-pathogen interaction pathways.

Conclusions: Our study contains an extensive sequencing and functional gene-annotation analysis of A. sativum L. The findings provide insights into the molecular basis of TFs, defensive genes, and a reference for future studies on the genetics and breeding of A. sativum L.