Development Of New Biotechnological Tools Research Articles

Microbial Biodiversity of Cryoconites in Palandöken Mountain and Culturing Cold-Adapted Enzyme Producers

Palandöken Mountain is among the highest and coldest ecosystems of Türkiye, and knowledge on its microbial diversity has not been adequately characterized. The aim of the present study is to expand the current understanding of microbial diversity in Palandöken Mountain. Sampling studies were carried out at in winter an altitude of 2400–2900 m. A next-generation sequencing (NGS) based culture-independent approach was employed for elucidating the community structure in the 8 cryoconite samples using 16S and 23S rRNA gene sequencing. In addition, biotechnologically important cold-adapted enzyme producers were isolated and identified by employing conventional culture-dependent studies. High-throughput sequencing analysis revealed the presence of 36 bacterial and 2 fungal phyla. The bacterial communities of 8 samples were dominated by Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Acidobacteria. The fungal communities were dominated by Ascomycota and Basidiomycota. 2900 different taxonomic group for bacterial diversity and 51 for fungal diversity were reported in the present study. Furthermore, 25 cold-adapted enzyme-producers were isolated and identified in the culture-dependent studies. Each selected isolate was capable of producing at least one of the enzymes amylase, protease, lipase, cellulase and xylanase isolates were assigned to Aspergillus, Bacillus, Cladosporium, Dioszegia, Flavobacterium, Fusarium, Holtermanniella, Naganishia, Polaromonas, Protomyces, Pseudomonas Rhodosporidiobolus, Vishniacozyma and Yarrowia genera. Consequently, this is the first geomicrobiological investigation that describes bacterial and fungal diversity of cryoconites from Palandöken Mountain using an NGS-based culture-independent approach. Additionally, cultured cold-adapted enzyme producer isolates of the present study have a valuable potential for the development of new biotechnological tools for various industrial applications.

Knowing Our Enemy in the Antimicrobial Resistance Era: Dissecting the Molecular Basis of Bacterial Defense Systems.

Bacteria and their phage adversaries are engaged in an ongoing arms race, resulting in the development of a broad antiphage arsenal and corresponding viral countermeasures. In recent years, the identification and utilization of CRISPR-Cas systems have driven a renewed interest in discovering and characterizing antiphage mechanisms, revealing a richer diversity than initially anticipated. Currently, these defense systems can be categorized based on the bacteria's strategy associated with the infection cycle stage. Thus, bacterial defense systems can degrade the invading genetic material, trigger an abortive infection, or inhibit genome replication. Understanding the molecular mechanisms of processes related to bacterial immunity has significant implications for phage-based therapies and the development of new biotechnological tools. This review aims to comprehensively cover these processes, with a focus on the most recent discoveries.

Proteomic Approach to Anemonia sulcata and Its Symbiont Symbiodinium spp. as New Source of Potential Biotechnological Applications and Climate Change Biomarkers.

Marine ecosystems are among the richest in terms of biodiversity, and at present, still remain largely unknown today. In the molecular biology era, several analyses have been conducted to unravel the biological processes in this ecosystem. These systems have provided biotechnological solutions to current problems, including the treatment of diseases, as well as for the development of new biotechnological tools with applications in biomedicine and/or agri-food. In addition, in the context of climate change and global warming, these studies become even more necessary for the development of molecular tools that allow a reliable follow-up of this situation to anticipate alterations and responses of bioindicator species and to create a database to prevent and predict the environmental and climatic changes before the damage is irreversible. Proteomics approaches have revealed their potential use to obtain the set of biological effectors that lead to the real biological station on a specific stage, the proteins. In addition, proteomics-based algorithms have allowed the discovery of proteins with new potential biotechnological applications from proteome data through "applied proteomics". In this project, the first proteome analysis of the sea anemone, Anemonia sulcata, and its symbiont has been developed. These organisms present a wide distribution sea ecosystem. In Spain, it is accepted as a fishing and aquaculture species. Moreover, Anemonia sulcate has a symbiotic relation with autotroph Dinoflagellates, Symbiodinium spp., that allows the study of its relation at the molecular level. For the first characterization of A. sulcata proteome, three independent biological replicates were used, and proteins were extracted and analyzed by LC-MS/MS, allowing the quantification of 325 proteins, 81 from Symbiodinium spp. proteins and 244 from A. sulcata proteins. These proteins were subjected to gene ontology categorization by Cellular Component, Molecular Function and Biological Process. These analyzes have allowed the identification of biomarkers of gene expression as potential powerful emerging diagnostic tools to identify and characterize the molecular drivers of climate change stresses and improve monitoring techniques. In addition, through the application of novel algorithms for the detection of bioactive compounds based on the analysis of molecules of marine origin, the proteome has allowed the identification of proteins with potential applications in the fields of biomedicine and agri-food.

Type I CRISPR-Cas-mediated microbial gene editing and regulation.

There are six major types of CRISPR-Cas systems that provide adaptive immunity in bacteria and archaea against invasive genetic elements. The discovery of CRISPR-Cas systems has revolutionized the field of genetics in many organisms. In the past few years, exploitations of the most abundant class 1 type I CRISPR-Cas systems have revealed their great potential and distinct advantages to achieve gene editing and regulation in diverse microorganisms in spite of their complicated structures. The widespread and diversified type I CRISPR-Cas systems are becoming increasingly attractive for the development of new biotechnological tools, especially in genetically recalcitrant microbial strains. In this review article, we comprehensively summarize recent advancements in microbial gene editing and regulation by utilizing type I CRISPR-Cas systems. Importantly, to expand the microbial host range of type I CRISPR-Cas-based applications, these structurally complicated systems have been improved as transferable gene-editing tools with efficient delivery methods for stable expression of CRISPR-Cas elements, as well as convenient gene-regulation tools with the prevention of DNA cleavage by obviating deletion or mutation of the Cas3 nuclease. We envision that type I CRISPR-Cas systems will largely expand the biotechnological toolbox for microbes with medical, environmental and industrial importance.

Purification, Characterization and Evaluation of the Antitumoral Activity of a Phospholipase A2 from the Snake Bothrops moojeni.

Nature presents a wide range of biomolecules with pharmacological potential, including venomous animal proteins. Among the protein components from snake venoms, phospholipases (PLA2) are of great importance for the development of new anticancer compounds. Thus, we aimed to evaluate the PLA2 anticancer properties from Bothrops moojeni venom. The crude venom was purified through three chromatographic steps, monitored by enzymatic activity and SDS-PAGE (12%). The purified PLA2 denominated BmPLA2 had its molecular mass and N-terminal sequence identified by mass spectrometry and Edman degradation, respectively. BmPLA2 was assayed against human epithelial colorectal adenocarcinoma cells (Caco-2), human rhabdomyosarcoma cells (RD) and mucoepidermoid carcinoma of the lung (NCI-H292), using human fibroblast cells (MRC-5) and microglia cells (BV-2) as a cytotoxicity control. BmPLA2 presented 13,836 Da and a 24 amino acid-residue homologue with snake PLA2, which showed a 90% similarity with other Bothrops moojeni PLA2. BmPLA2 displayed an IC50 of 0.6 µM against Caco-2, and demonstrated a selectivity index of 1.85 (compared to MRC-5) and 6.33 (compared to BV-2), supporting its selectivity for cancer cells. In conclusion, we describe a new acidic phospholipase, which showed antitumor activity and is a potential candidate in the development of new biotechnological tools.

In planta RNAi approach targeting three M. incognita effector genes disturbed the process of infection and reduced plant susceptibility

Meloidogyne incognita is the most economically important species of the root-knot nematode complex causing damage to several crops worldwide. During parasitism in host plants, M. incognita secretes several effector proteins to suppress the plant immune system, manipulate the plant cell cycle, and promote parasitism. Several effector proteins have been identified, but their relationship with plant parasitism by M. incognita has not been fully confirmed. Herein, the Minc01696, Minc00344, and Minc00801 putative effector genes were evaluated to assess their importance during soybean and Nicotiana tabacum parasitism by M. incognita. For this study, we used in planta RNAi technology to overexpress dsRNA molecules capable of producing siRNAs that target and downregulate these nematode effector genes. Soybean composite roots and N. tabacum lines were successfully generated, and susceptibility level to M. incognita was evaluated. Consistently, both transgenic soybean roots and transgenic N. tabacum lines carrying the RNAi strategy showed reduced susceptibility to M. incognita. The number of galls per plant and the number of egg masses per plant were reduced by up to 85% in transgenic soybean roots, supported by the downregulation of effector genes in M. incognita during parasitism. Similarly, the number of galls per plant, the number of egg masses per plant, and the nematode reproduction factor were reduced by up to 83% in transgenic N. tabacum lines, which was also supported by the downregulation of the Minc00801 effector gene during parasitism. Therefore, our data indicate that all three effector genes can be a target in the development of new biotechnological tools based on the RNAi strategy in economically important crops for M. incognita control.

Bioprospecting Kluyveromyces marxianus as a Robust Host for Industrial Biotechnology.

Kluyveromyces marxianus is an emerging non-conventional food-grade yeast that is generally isolated from diverse habitats, like kefir grain, fermented dairy products, sugar industry sewage, plants, and sisal leaves. A unique set of beneficial traits, such as fastest growth, thermotolerance, and broad substrate spectrum (i.e., hemi-cellulose hydrolysates, xylose, l-arabinose, d-mannose, galactose, maltose, sugar syrup molasses, cellobiose, and dairy industry) makes this yeast a particularly attractive host for applications in a variety of food and biotechnology industries. In contrast to Saccharomyces cerevisiae, most of the K. marxianus strains are apparently Crabtree-negative or having aerobic-respiring characteristics, and unlikely to endure aerobic alcoholic fermentation. This is a desirable phenotype for the large-scale biosynthesis of products associated with biomass formation because the formation of ethanol as an undesirable byproduct can be evaded under aerobic conditions. Herein, we discuss the current insight into the potential applications of K. marxianus as a robust yeast cell factory to produce various industrially pertinent enzymes, bioethanol, cell proteins, probiotic, fructose, and fructo-oligosaccharides, and vaccines, with excellent natural features. Moreover, the biotechnological improvement and development of new biotechnological tools, particularly CRISPR–Cas9-assisted precise genome editing in K. marxianus are delineated. Lastly, the ongoing challenges, concluding remarks, and future prospects for expanding the scope of K. marxianus utilization in modern biotechnology, food, feed, and pharmaceutical industries are also thoroughly vetted. In conclusion, it is critical to apprehend knowledge gaps around genes, metabolic pathways, key enzymes, and regulation for gaining a complete insight into the mechanism for producing relevant metabolites by K. marxianus.

Beta-1,3-glucanase inhibitors in Brazilian brown seaweed.

Beta-1,3-glucanases are enzymes that hydrolyze beta-1,3-glucans, and they are essential for the metabolism of seaweed, plants and fungi. These enzymes also participate in the digestion of herbivore and fungivore animals. Because of the importance of these enzymes in insects, beta-1,3-glucanase inhibitors may be used for the development of new control strategies against agricultural pests and disease vectors. Beta-1,3-glucanase inhibitors have been described in the brown seaweed Laminaria cichorioides, but were never recorded in Brazilian seaweed species. We evaluated the presence of beta-1,3-glucanase inhibitors in samples of Padina gymnospora, Dictyota sp., Colpomenia sinuosa, and Lobophora sp., collected in Arraial d'Ajuda (Bahia). Ethanolic or buffer extracts were used in inhibition tests against the beta-1,3-glucanase of Trichoderma sp. Extracts in buffer showed no inhibition, but ethanolic extracts from all species showed different extents of inhibition. Samples from Dictyota sp. and P. gymnospora showed inhibitions above 75% (absolute ethanol) or 50% (ethanol 50%). In summary, extraction with absolute ethanol resulted in better inhibitions, and P. gymnospora showed the higher inhibitions. Brazilian seaweed may be good sources of beta-1,3-glucanase inhibitors for biochemical and physiological studies of these enzymes. Besides that, these molecules show potential for the development of new biotechnological tools for insect control.

Insights obtained using different modules of the cotton uceA1.7 promoter.

The structure of the cotton uceA1.7 promoter and its modules was analyzed; the potential of their key sequences has been confirmed in different tissues, proving to be a good candidate for the development of new biotechnological tools. Transcriptional promoters are among the primary genetic engineering elements used to control genes of interest (GOIs) associated with agronomic traits. Cotton uceA1.7 was previously characterized as a constitutive promoter with activity higher than that of the constitutive promoter from the Cauliflower mosaic virus (CaMV) 35S gene in various plant tissues. In this study, we generated Arabidopsis thaliana homozygous events stably overexpressing the gfp reporter gene driven by different modules of the uceA1.7 promoter. The expression level of the reporter gene in different plant tissues and the transcriptional stability of these modules was determined compared to its full-length promoter and the 35S promoter. The full-length uceA1.7 promoter exhibited higher activity in different plant tissues compared to the 35S promoter. Two modules of the promoter produced a low and unstable transcription level compared to the other promoters. The other two modules rich in cis-regulatory elements showed similar activity levels to full-length uceA1.7 and 35S promoters but were less stable. This result suggests the location of a minimal portion of the promoter that is required to initiate transcription properly (the core promoter). Additionally, the full-length uceA1.7 promoter containing the 5'-untranslated region (UTR) is essential for higher transcriptional stability in various plant tissues. These findings confirm the potential use of the full-length uceA1.7 promoter for the development of new biotechnological tools (NBTs) to achieve higher expression levels of GOIs in, for example, the root or flower bud for the efficient control of phytonematodes and pest-insects, respectively, in important crops.

The arms race between bacteria and their phage foes.

Bacteria are under immense evolutionary pressure from their viral invaders-bacteriophages. Bacteria have evolved numerous immune mechanisms, both innate and adaptive, to cope with this pressure. The discovery and exploitation of CRISPR-Cas systems have stimulated a resurgence in the identification and characterization of anti-phage mechanisms. Bacteriophages use an extensive battery of counter-defence strategies to co-exist in the presence of these diverse phage defence mechanisms. Understanding the dynamics of the interactions between these microorganisms has implications for phage-based therapies, microbial ecology and evolution, and the development of new biotechnological tools. Here we review the spectrum of anti-phage systems and highlight their evasion by bacteriophages.

When spider and snake get along: Fusion of a snake disintegrin with a spider phospholipase D to explore their synergistic effects on a tumor cell

Venoms of spiders and snakes contain toxins extremely active and, thus, provide a natural source for the development of new biotechnological tools. Among the diversity of toxins present in the venom of spiders from genus Loxosceles, the phospholipases D (PLDs) show high hydrolytic activity upon lysophosphatidylcholine (LPC) and sphingomyelin (SM), generating bioactive phospholipids such as cyclic phosphatidic acid (cPA). Since this mediator has been shown to play a major role in complex signaling pathways, including inhibition of tumor cells, the PLDs may hold the key to learn how toxins could be used for therapeutic purposes. However, the strong platelet aggregation of PLDs and their lack of selectivity impose a major limitation. On the other hand, disintegrins present in the venoms of Viperidae snakes are a potent inhibitor of platelet aggregation and possess high affinity and specificity to molecules called integrins that are highly expressed in some tumor cells, such as murine melanoma B16F10. Therefore, disintegrins might be suitable molecules to carry the PLDs to the malignant cells, so both toxins may work synergistically to eliminate these cells. Thus, in this work, a recombinant PLD from Loxosceles gaucho spider was recombinantly fused to a disintegrin from Echis carinatus snake to form a hybrid toxin called Rechistatin. This recombinant toxin was successfully expressed in bacteria, showed binding activity in B16F10 murine melanoma cells and exerted a synergistic cytotoxicity effect on these cells. Therefore, the approach presented in this work may represent a new strategy to explore new potential applications for spider PLDs.

New Biotechnological Tools for the Genetic Improvement of Major Woody Fruit Species.

The improvement of woody fruit species by traditional plant breeding techniques has several limitations mainly caused by their high degree of heterozygosity, the length of their juvenile phase and auto-incompatibility. The development of new biotechnological tools (NBTs), such as RNA interference (RNAi), trans-grafting, cisgenesis/intragenesis, and genome editing tools, like zinc-finger and CRISPR/Cas9, has introduced the possibility of more precise and faster genetic modifications of plants. This aspect is of particular importance for the introduction or modification of specific traits in woody fruit species while maintaining unchanged general characteristics of a selected cultivar. Moreover, some of these new tools give the possibility to obtain transgene-free modified fruit tree genomes, which should increase consumer's acceptance. Over the decades biotechnological tools have undergone rapid development and there is a continuous addition of new and valuable techniques for plant breeders. This makes it possible to create desirable woody fruit varieties in a fast and more efficient way to meet the demand for sustainable agricultural productivity. Although, NBTs have a common goal i.e., precise, fast, and efficient crop improvement, individually they are markedly different in approach and characteristics from each other. In this review we describe in detail their mechanisms and applications for the improvement of fruit trees and consider the relationship between these biotechnological tools and the EU biosafety regulations applied to the plants and products obtained through these techniques.

Many Activities, One Structure: Functional Plasticity of Ribozyme Folds.

Catalytic RNAs, or ribozymes, are involved in a number of essential biological processes, such as replication of RNA genomes and mobile genetic elements, RNA splicing, translation, and RNA degradation. The function of ribozymes requires the formation of active sites decorated with RNA functional groups within defined three-dimensional (3D) structures. The genotype (sequence) of RNAs ultimately determines what 3D structures they adopt (as a function of their environmental conditions). These 3D structures, in turn, give rise to biochemical activity, which can further elaborate them by catalytic rearrangements or association with other molecules. The fitness landscape of a non-periodic linear polymer, such as RNA, relates its primary structure to a phenotype. Two major challenges in the analysis of ribozymes is to map all possible genotypes to their corresponding catalytic activity (that is, to determine their fitness landscape experimentally), and to understand whether their genotypes and three-dimensional structures can support multiple different catalytic functions. Recently, the combined results of experiments that employ in vitro evolution methods, high-throughput sequencing and crystallographic structure determination have hinted at answers to these two questions: while the fitness landscape of ribozymes is rugged, meaning that their catalytic activity cannot be optimized by a smooth trajectory in sequence space, once an RNA achieves a stable three-dimensional fold, it can be endowed with distinctly different biochemical activities through small changes in genotype. This functional plasticity of highly structured RNAs may be particularly advantageous for the adaptation of organisms to drastic changes in selective pressure, or for the development of new biotechnological tools.

Anthocyanin Biosynthesis Regulation in the Fruit of Citrus sinensis cv. Tarocco

The differential expression profiling of the fruit-specific genes between a blood orange (Citrus sinensis cv. Tarocco) and a blonde orange (Citrus sinensis cv. twenty-first century navel orange) was investigated by fruit transcriptome sequencing. The relative expression folds for Cs6g17570 and Cs5g31400 were significantly greater in the “Tarocco” fruits, but Cs9g04810 transcripts had no significant differences between two accessions by digital gene expression profiling. In particular, R2R3-MYB, bHLH-type, and WD40-repeat proteins (encoded by Cs6g17570, Cs5g31400, and Cs9g04810) were transcription factors with each conserved domain and interacted in vitro and in vivo. Moreover, the dihydroflavonol 4-reductase (DFR), a key enzyme involved into anthocyanin biosynthesis pathway, had greater transcription levels in Tarocco fruit and also in the transgenic Arabidopsis thaliana by Cs6g17570 ectopic expression. Furthermore, Cs6g17570 and Cs5g31400 in Tarroco were upregulated by cold induction and then had similar expression profiling during cold storage. In conclusion, Cs6g17570, Cs5g31400, and Cs9g04810 encoding proteins might be involved into anthocyanin regulation in Tarocco by MYB-bHLH-WD40 complex as well as in other fruit trees. Studies on the anthocyanin regulatory mechanism will aid in development of new biotechnological tools for Citrus breeding.

Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway

Flavonoids are secondary metabolites involved in several aspects of plant development and defence. They colour fruits and flowers, favouring seed and pollen dispersal, and contribute to plant adaptation to environmental conditions such as cold or UV stresses, and pathogen attacks. Because they affect the quality of flowers (for horticulture), fruits and vegetables, and their derivatives (colour, aroma, stringency, etc.), flavonoids have a high economic value. Furthermore, these compounds possess pharmaceutical properties extremely attractive for human health. Thanks to easily detectable mutant phenotypes, such as modification of petal pigmentation and seeds exhibiting transparent testa, the enzymes involved in the flavonoid biosynthetic pathway have been characterized in several plant species. Conserved features as well as specific differences have been described. Regulation of structural gene expression appears tightly organized in a spatial and temporal way during plant development, and is orchestrated by a ternary complex involving transcription factors from the R2R3-MYB, basic helix-loop-helix (bHLH), and WD40 classes. This MYB-bHLH-WD40 (MBW) complex regulates the genes that encode enzymes specifically involved in the late steps of the pathway leading to the biosynthesis of anthocyanins and condensed tannins. Although several genes encoding transcription factors from these three families have been identified, many gaps remain in our understanding of the regulation of this biosynthetic pathway, especially about the respective roles of bHLH and WD40 proteins. A better knowledge of the regulatory mechanisms of the flavonoid pathway is likely to favour the development of new biotechnological tools for the generation of value-added plants with optimized flavonoid content.

Les biothérapies immunomodulatrices du futur : quelles perspectives ?

During the last decade, new biotherapies have been developed for the treatment of systemic autoimmune diseases. These new approaches are based on a better understanding of the auto-immune response. Targets of these new treatments are the main steps of the immune response. These new treatments include: B lymphocytes inhibitors, inhibitors of the costimulation between antigen-presenting cells and T lymphocytes, T lymphocytes inhibitors, B and T lymphocytes inhibitors, modulators of the cytokine network, complement system inhibitors, and chemokines and integrins inhibitors. Advances in molecular biology, genetics and immunology have accelerated our understanding of auto-immune diseases, and allow identification of new targets. Development of new biotechnological tools with longer half-lives and increased affinity is an important next step. In spite of these exciting new perspectives, a systematic risk/benefit evaluation of these new treatments appears mandatory because immunity plays a key role in the control of infectious and tumor diseases. Further research is also needed to evaluate the cost/effectiveness ratio of these new therapies.

Molecular cytogenetics in translational oncology: when chromosomes meet genomics

The discovery of the genetic changes that contribute to cellular neoplastic transformation is one of the major aims in oncological research. Chromosome rearrangements account for a large part of these initiating mutations that, resulting in gene deregulation, are the main target of molecular cytogenetics. Cytogenetics, based in reasoned genomic and biological questions and supported by the development of new biotechnological tools, is a powerful discipline that is continuously generating pieces of information that have immediate translation as reagents for diagnosis and useful research data. The present review presents a summary of the major cytogenetic findings that already have a clear role in clinical oncology because of their use as diagnostic markers, as indicators of molecular therapy suitability or both. We also present an updated description of the molecular cytogenetics tools that have included genomic advances in their most recent releases: multicolour fluorescence in situ hybridisation methods (e.g. SKY karyotyping) and array-based comparative genomic hybridisation (arrayCGH).

Perspectives on biotechnological applications of archaea.

Many archaea colonize extreme environments. They include hyperthermophiles, sulfur-metabolizing thermophiles, extreme halophiles and methanogens. Because extremophilic microorganisms have unusual properties, they are a potentially valuable resource in the development of novel biotechnological processes. Despite extensive research, however, there are few existing industrial applications of either archaeal biomass or archaeal enzymes. This review summarizes current knowledge about the biotechnological uses of archaea and archaeal enzymes with special attention to potential applications that are the subject of current experimental evaluation. Topics covered include cultivation methods, recent achievements in genomics, which are of key importance for the development of new biotechnological tools, and the application of wild-type biomasses, engineered microorganisms, enzymes and specific metabolites in particular bioprocesses of industrial interest.