Journal of Genetic Engineering and Biotechnology

Exopolysaccharides (EPSs) từ vi sinh vật là các biopolymer tự nhiên vô hại quan trọng, được sử dụng trong các ứng dụng bao gồm dược phẩm, nutraceuticals và thực phẩm chức năng, mỹ phẩm và thuốc trừ sâu. Nhiều loại vi sinh vật có khả năng tổng hợp và bài tiết EPSs với các tính chất hóa học và cấu trúc khiến chúng phù hợp với một số ứng dụng quan trọng. Vi sinh vật bài tiết EPS ra bên ngoài màng tế bào của chúng, dưới dạng chất nhờn hoặc “thạch” vào môi trường ngoại bào. Những vi sinh vật sản xuất EPS này rất phổ biến và có thể được phân lập từ các môi trường nước và đất, chẳng hạn như nước ngọt, nước biển, nước thải và đất. Chúng cũng đã được phân lập từ những môi trường cực đoan như suối nước nóng, nước lạnh, các môi trường ưa muối và đầm lầy muối. Gần đây, EPS vi sinh vật đã thu hút sự quan tâm cho các ứng dụng như tác nhân kết tụ sinh học môi trường nhờ tính chất phân hủy được và không độc hại của chúng. Tuy nhiên, cần có thêm nỗ lực để sản xuất EPS vi sinh vật một cách hiệu quả về chi phí và quy mô công nghiệp. Bài đánh giá này tập trung vào các exopolysaccharides thu được từ nhiều vi sinh vật ưa cực, quá trình tổng hợp của chúng và tối ưu hóa sản xuất để cải thiện chi phí và năng suất. Chúng tôi cũng đã khám phá vai trò và ứng dụng của chúng trong các tương tác giữa nhiều sinh vật khác nhau.

Hepatitis E is a liver disease caused by the pathogen hepatitis E virus (HEV). The largest polyprotein open reading frame 1 (ORF1) contains a nonstructural Y-domain region (YDR) whose activity in HEV adaptation remains uncharted. The specific role of disordered regions in several nonstructural proteins has been demonstrated to participate in the multiplication and multiple regulatory functions of the viruses. Thus, intrinsic disorder of YDR including its structural and functional annotation was comprehensively studied by exploiting computational methodologies to delineate its role in viral adaptation. Based on our findings, it was evident that YDR contains significantly higher levels of ordered regions with less prevalence of disordered residues. Sequence-based analysis of YDR revealed it as a “dual personality” (DP) protein due to the presence of both structured and unstructured (intrinsically disordered) regions. The evolution of YDR was shaped by pressures that lead towards predominance of both disordered and regularly folded amino acids (Ala, Arg, Gly, Ile, Leu, Phe, Pro, Ser, Tyr, Val). Additionally, the predominance of characteristic DP residues (Thr, Arg, Gly, and Pro) further showed the order as well as disorder characteristic possessed by YDR. The intrinsic disorder propensity analysis of YDR revealed it as a moderately disordered protein. All the YDR sequences consisted of molecular recognition features (MoRFs), i.e., intrinsic disorder-based protein–protein interaction (PPI) sites, in addition to several nucleotide-binding sites. Thus, the presence of molecular recognition (PPI, RNA binding, and DNA binding) signifies the YDR’s interaction with specific partners, host membranes leading to further viral infection. The presence of various disordered-based phosphorylation sites further signifies the role of YDR in various biological processes. Furthermore, functional annotation of YDR revealed it as a multifunctional-associated protein, due to its susceptibility in binding to a wide range of ligands and involvement in various catalytic activities. As DP are targets for regulation, thus, YDR contributes to cellular signaling processes through PPIs. As YDR is incompletely understood, therefore, our data on disorder-based function could help in better understanding its associated functions. Collectively, our novel data from this comprehensive investigation is the first attempt to delineate YDR role in the regulation and pathogenesis of HEV.

Since the index case was reported in China, COVID-19 has led to the death of at least 4 million people globally. Although there are some vaccine cocktails in circulation, the emergence of more virulent variants of SARS-CoV-2 may make the eradication of COVID-19 more difficult. Nsp16 is an S-adenosyl-L-Methionine-dependent methyltransferase that plays an important role in SARS-CoV-2 viral RNA cap formation—a crucial process that confers viral stability and prevents virus detection by cell innate immunity mechanisms. This unique property makes nsp16 a promising molecular target for COVID-19 drug design. Thus, this study aimed to identify potent phytocompounds that can effectively inhibit SARS-CoV-2 nsp16. We performed in silico pharmacokinetic screening and molecular docking studies using 100 phytocompounds—isolated from fourteen Nigerian plants—as ligands and nsp16 (PDB: 6YZ1) as the target. We found that only 59 phytocompounds passed the drug-likeness analysis test. However, after the docking analysis, only six phytocompounds (oxopowelline, andrographolide, deacetylbowdensine, 11, 12-dimethyl sageone, sageone, and quercetin) isolated from four Nigerian plants (Crinum jagus, Andrographis paniculata, Sage plants (Salvia officinalis L.), and Anacardium occidentale) showed good binding affinity with nsp16 at its active site with docking score ranging from − 7.9 to − 8.4 kcal/mol. Our findings suggest that the six phytocompounds could serve as therapeutic agents to prevent viral survival and replication in cells. However, further studies on the in vitro and in vivo inhibitory activities of these 6 hit phytocompounds against SARS-CoV-2 nsp16 are needed to confirm their efficacy and dose.

Amoebiasis, being endemic worldwide, is the second leading cause of parasite-associated morbidity and mortality after malaria. The human parasite Entamoeba histolytica is responsible for the disease. Metronidazole is considered as the gold standard for the treatment of amoebiasis, but this antibiotic is carcinogenic and the development of antibiotic resistance against E. histolytica is a major health concern. Chromosome segregation is irregular in this parasite due to the absence of a few cell cycle checkpoint proteins. Anaphase-promoting complex (APC/C or cyclosome) is an E3 ubiquitin ligase that synchronizes chromosome segregation and anaphase progression via the ubiquitin-proteasome system. Proteasome is considered to be an attractive drug target for protozoan parasites. For the present study, EhApc11 from E. histolytica, a homologue of Apc11 in humans, is selected for elucidating its structural and functional aspects by detailed in silico analysis and molecular methods. Its physicochemical characteristics, identification of probable interactors, 3D model and quality analysis are done using standard bioinformatics tools. cDNA sequence of EhAPC11 has been further cloned for molecular characterization. Conserved domain analysis revealed that EhApc11 belongs to the RING (really interesting new gene) superfamily and has ligand binding capacity. Expression study in Escherichia coli BL21 (DE3) revealed that the molecular weight of glutathione S-transferase (GST)-tagged protein is ~ 36 kDa. EhApc11 is a hydrophilic, thermostable, extracellular protein with potent antigenicity. The study will serve as a groundwork for future in-depth analysis regarding the validation of protein-protein interaction of EhApc11 with its substrates identified by STRING analysis and the potential of EhApc11 to serve as an anti-amoebic drug target.

Dalbergia sissoo (shisham), an important multipurpose tree native to the Indian subcontinent and also planted in other countries, has been afflicted with large scale mortality in all age groups due to wilt disease, causing huge economic losses. Fusarium solani f. sp. dalbergiae (Fsd) has been identified as one of the causal organisms for wilt disease in D. sissoo. One of the approaches of disease resistance studies involves co-cultivation of trees and pathogens under controlled conditions to screen resistant tree genotypes. A gnotobiotic condition, where the pathogen is known, enables accurate screening of disease-resistant genotypes. In the present study, ten genotypes of D. sissoo were cloned in vitro and evaluated against two strains of Fsd in a dual culture setup under gnotobiotic conditions with an objective to identify resistant genotypes of D. sissoo against Fsd. Callus and plantlets of ten genotypes of host plant multiplied in vitro were inoculated with conidial suspension of two strains of Fsd at three concentrations; 1 × 101, 1 × 103, and 1 × 105 conidia/ml. Gnotobiotic evaluation of dual culture setup shows variations among genotypes in their response towards in vitro Fsd infection; and two genotypes (14 and 66) exhibited resistance against Fsd strains. Callus of genotypes 14 and 66 significantly restricted the fungal mycelium growth whereas callus of remaining genotypes was completely infested by Fsd mycelium within 9 days. Similarly, plantlets of genotype 14 and 66 had lesser disease severity and remained green and had fewer necrotic lesions in roots whereas plantlets of the remaining eight genotypes died within 15 days. Gnotobiotic evaluation of callus and plantlets of ten genotypes of D. sissoo against Fsd strains has reduced time and space otherwise required for field trials. Genetic variations amongst the genotypes resulted in varying responses towards virulent Fsd strains and only two out of ten genotypes showed promising resistant characteristics. In dual culture setup, both callus and plantlets of the same genotypes responded similarly against Fsd strains, which signify that in vitro screening can be used as an indirect selection method for disease resistance.

The crucial factor in the production of bio-fuels is the choice of potent microorganisms used in fermentation processes. Despite the evolving trend of using bacteria, yeast is still the primary choice for fermentation. Molecular characterization of many genes from baker’s yeast (Saccharomyces cerevisiaea), and fission yeast (Schizosaccharomyces pombe), have improved our understanding in gene structure and the regulation of its expression. This in silico study was done with the aim of analyzing the promoter regions, transcription start site (TSS), and CpG islands of genes encoding for alcohol production in S. cerevisiaea S288C and S. pombe 972h-. The analysis revealed the highest promoter prediction scores (1.0) were obtained in five sequences (AAD4, SFA1, GRE3, YKL071W, and YPR127W) for S. cerevisiaea S288C TSS while the lowest (0.8) were found in three sequences (AAD6, ADH5, and BDH2). Similarly, in S. pombe 972h-, the highest (0.99) and lowest (0.88) prediction scores were obtained in five (Adh1, SPBC8E4.04, SPBC215.11c, SPAP32A8.02, and SPAC19G12.09) and one (erg27) sequences, respectively. Determination of common motifs revealed that S. cerevisiaea S288C had 100% coverage at MSc1 with an E value of 3.7e−007 while S. pombe 972h- had 95.23% at MSp1 with an E value of 2.6e+002. Furthermore, comparison of identified transcription factor proteins indicated that 88.88% of MSp1 were exactly similar to MSc1. It also revealed that only 21.73% in S. cerevisiaea S288C and 28% in S. pombe 972h- of the gene body regions had CpG islands. A combined phylogenetic analysis indicated that all sequences from both S. cerevisiaea S288C and S. pombe 972h- were divided into four subgroups (I, II, III, and IV). The four clades are respectively colored in blue, red, green, and violet. This in silico analysis of gene promoter regions and transcription factors through the actions of regulatory structure such as motifs and CpG islands of genes encoding alcohol production could be used to predict gene expression profiles in yeast species.

The green peach aphid, Myzus persicae Sulzer, and the bean aphid, Aphis fabae Scopoli (both Hemiptera: Aphididae), are serious pests of greenhouse vegetable crops in Iraq and other regions of the globe. In this study, two morphological identical isolates (AA80 and AA82) of the entomopathogenic fungus Clonostachys rosea Schroers (Hypocreales: Bionectriaceae) from Iraq were isolated and characterized with phylogenetic analysis based on the internal transcribed spacer (ITS) region. The efficacy of C. rosea against M. persicae and A. fabae was previously unknown. In the laboratory bioassays, mortality of adult M. persicae and A. fabae caused by both C. rosea isolates varied according to conidial concentrations, with complete mortality occurring at 1 × 109 conidia ml−1 10 day post treatment. For M. persicae, LC50 values of AA80 and AA80 isolates were 3.6 × 106 and 3.8 × 106 conidia ml−1. For A. fabae, LC50 values of AA80 and AA80 isolates were 4.5 × 106 and 4.35 × 106 conidia ml−1. Infection by both fungal isolates at LC50 values reduced total fecundity of the treated aphids by 20% when compared to the untreated aphids. The results from laboratory bioassays showed that C. rosea has potential as a biological control agent of M. persicae and A. fabae which is crucial for ecofriendly biopesticide development. However, further field and greenhouse studies are required for mass production.

The Capparaceae family is commonly recognized as a caper, while Cleomaceae represents one of small flowering family within the order Brassicales. Earlier, Cleomaceae was included in the family Capparaceae; then, it was moved to a distinct family after DNA evidence. Variation in habits and a bewildering array of floral and fruit forms contributed to making Capparaceae a “trash-basket” family in which many unrelated plants were placed. Indeed, family Capparaceae and Cleomaceae are in clear need of more detailed systematic revision. Here, in the present study, the morphological characteristics and the ecological distribution as well as the genetic diversity analysis among the twelve species of both Capparaceae and Cleomaceae have been determined. The genetic analysis has been checked using 15 ISSR, 30 SRAP, and 18 ISTR to assess the systematic knots between the two families. In order to detect the molecular phylogeny, a comparative analysis of the three markers was performed based on the exposure of discriminating capacity, efficiency, and phylogenetic heatmap. Our results indicated that there is a morphological and ecological variation between the two families. Moreover, the molecular analysis confirmed that ISTR followed by SRAP markers has superior discriminating capacity for describing the genetic diversity and is able to simultaneously distinguish many polymorphic markers per reaction. Indeed, both the PCA and HCA data have drawn a successful annotation relationship in Capparaceae and Cleome species to evaluate whether the specific group sort individual or overlap groups. The outcomes of the morphological and ecological characterization along with the genetic diversity indicated an insight solution thorny interspecies in Cleome and Gynandropsis genera as a distinct family (Cleomaceae) and the other genera (Capparis, Cadaba, Boscia, and Maerua) as Capparaceae. Finally, we recommended further studies to elucidate the systematic position of Dipterygium glaucum.

Potato (Solanum tuberosum L.) is one of the most important food crops in the world. Pathogens remain as one of the major constraints limiting potato productivity. Thus, understanding of gene regulation mechanism of pathogenesis-related genes such as glucan endo-1,3-beta-glucosidase is a foundation for genetic engineering of potato for disease resistance and reduces the use of fungicides. In the present study, 19 genes were selected and attempts were made through in silico methods to identify and characterize the promoter regions, regulatory elements, and CpG islands of glucan endo-1,3-beta-glucosidase gene in Solanum tuberosum cultivar DM 1-3 516 R44. The current analysis revealed that single transcription start sites (TSSs) were present in 12/19 (63.2%) of promoter regions analyzed. The predictive score at a cutoff value of 0.8 for the majority (84.2%) of the promoter regions ranged from 0.90 to 1.00. The locations for 42% of the TSSs were below −500 bp relative to the start codon (ATG). MβGII was identified as the common promoter motif for 94.4% of the genes with an E value of 3.5e−001. The CpG analysis showed low CpG density in the promoter regions of most of the genes except for gene ID102593331 and ID: 102595860. The number of SSRs per gene ranged from 2 to 9 with repeat lengths of 2 to 6 bp. Evolutionary distances ranged from 0.685 to 0.770 (mean = 0.73), demonstrating narrower genetic diversity range. Phylogeny was inferred using the UPGMA method, and gene sequences from different species were found to be clustered together. In silico identified regulatory elements in promoter regions will contribute to our understanding of the regulatory mechanism of glucan endo-1,3-beta-glucosidase genes and provide a promising target for genetic engineering to improve disease resistance in potatoes.