Biotechnological Applications Research Articles

The fnr-like mutants confer isoxaben tolerance by initiating mitochondrial retrograde signalling.

Isoxaben is a pre-emergent herbicide used to control broadleaf weeds. While the phytotoxic mechanism is not completely understood, isoxaben interferes with cellulose synthesis. Certain mutations in cellulose synthase complex proteins can confer isoxaben tolerance; however, these mutations can cause compromised cellulose synthesis and perturbed plant growth, rendering them unsuitable as herbicide tolerance traits. We conducted a genetic screen to identify new genes associated with isoxaben tolerance by screening a selection of Arabidopsis thaliana T-DNA mutants. We found that mutations in a FERREDOXIN-NADP(+) OXIDOREDUCTASE-LIKE (FNRL) gene enhanced tolerance to isoxaben, exhibited as a reduction in primary root stunting, reactive oxygen species accumulation and ectopic lignification. The fnrl mutant did not exhibit a reduction in cellulose levels following exposure to isoxaben, indicating that FNRL operates upstream of isoxaben-induced cellulose inhibition. In line with these results, transcriptomic analysis revealed a highly reduced response to isoxaben treatment in fnrl mutant roots. The fnrl mutants displayed constitutively induced mitochondrial retrograde signalling, and the observed isoxaben tolerance is partially dependent on the transcription factor ANAC017, a key regulator of mitochondrial retrograde signalling. Moreover, FNRL is highly conserved across all plant lineages, implying conservation of its function. Notably, fnrl mutants did not show a growth penalty in shoots, making FNRL a promising target for biotechnological applications in breeding isoxaben tolerance in crops.

Production of bacteriocin-like inhibitory substance (BLIS) by Staphylococcus spp. isolates from dogs.

In the present study, 39 canine isolates of Staphylococcus spp. were tested for antimicrobial substance (AMS) production. Seven AMS producers were identified, whose products exhibited a non-acidic character and a proteinaceous nature, therefore being considered bacteriocin-like inhibitory substances (BLIS). The producer strains of BLIS P1, P16 and I3 showed a broad spectrum of antimicrobial activity. Human, veterinary and plant pathogens, such as Listeria monocytogenes, Klebsiella pneumoniae, Staphylococcus spp. and Clavibacter michiganensis, were among the inhibited micro-organisms, suggesting the potential biotechnological application of these peptides. MALDI-TOF mass spectrometry and 16S rDNA sequencing identified the producer strains of BLIS P1, P16 and I3 as Staphylococcus pseudintermedius P1, Staphylococcus schleiferi P16 and Staphylococcus pseudintermedius I3. The plasmid profile of these strains suggests that the BLIS production is linked to biosynthetic genes located on plasmids. PCR analyses revealed that BLIS P1, P16 and I3 are different from 11 staphylococcins already described in the literature and that their genomic DNAs do not carry the most prevalent staphylococcal enterotoxin genes. The highest levels of BLIS production were achieved after 18-24h of growth of the producer strains in TSB medium. Moreover, BLIS P1 and I3 exhibited high resistance to temperature and pH variations, and BLIS P16 maintained 100% of its activity in almost all conditions tested. The characteristics associated with BLIS P1, P16 and I3 described in this work encourage further investigation of these substances, in addition to this study being the first report of BLIS production by a strain of S. schleiferi.

Discovery of Lacto-N-Biosidases and a Novel N-Acetyllactosaminidase Activity in the CAZy Family GH20: Functional Diversity and Structural Insights.

The glycoside hydrolase family 20 (GH20) predominantly features N-acetylhexosaminidases (EC 3.2.1.52), with only few known lacto-N-biosidases (EC 3.2.1.140; LNBases). LNBases catalyze the degradation of lacto-N-tetraose (LNT), a prominent component of human milk oligosaccharides, thereby supporting a healthy infant gut microbiome development. We investigated GH20 diversity to discover novel enzymes that release disaccharides such as lacto-N-biose (LNB). Our approach combined peptide clustering, sequence analysis, and 3D structure model evaluation to assess active site topologies, focusing on the presence of a subsite -2. Five LNBases were active on pNP-LNB and four showed activity on LNT. One enzyme displayed activity on both pNP-LacNAc and pNP-LNB, establishing the first report of N-acetyllactosaminidase (LacNAcase) activity. Exploration of this enzyme cluster led to the identification of four additional enzymes sharing this dual substrate specificity. Comparing the determined crystal structure of a specific LNBase (TrpyGH20) and the first crystal structure of an enzyme with dual LacNAcase/LNBase activity (TrdeGH20) revealed a highly conserved subsite -1, common to GH20 enzymes, while the -2 subsites varied significantly. TrdeGH20 had a wider subsite -2, accommodating Gal with both β1,4- and β1,3-linkages to the GlcNAc in subsite -1. Biotechnological applications of these enzymes may include structural elucidation of complex carbohydrates and glycoengineering.

Deep quantification of substrate turnover defines protease subsite cooperativity.

Substrate specificity determines protease functions in physiology and in clinical and biotechnological applications, yet quantitative cleavage information is often unavailable, biased, or limited to a small number of events. Here, we develop qPISA (quantitative Protease specificity Inference from Substrate Analysis) to study Dipeptidyl Peptidase Four (DPP4), a key regulator of blood glucose levels. We use mass spectrometry to quantify >40,000 peptides from a complex, commercially available peptide mixture. By analyzing changes in substrate levels quantitatively instead of focusing on qualitative product identification through a binary classifier, we can reveal cooperative interactions within DPP4's active pocket and derive a sequence motif that predicts activity quantitatively. qPISA distinguishes DPP4 from the related C. elegans DPF-3 (a DPP8/9-orthologue), and we relate the differences to the structural features of the two enzymes. We demonstrate that qPISA can direct protein engineering efforts like the stabilization of GLP-1, a key DPP4 substrate used in the treatment of diabetes and obesity. Thus, qPISA offers a versatile approach for profiling protease and especially exopeptidase specificity, facilitating insight into enzyme mechanisms and biotechnological and clinical applications.

Phytochemical Associes of Fungal Endophytes in Nigeria Riverine Mangrove Ecosystem

The interaction between endophytic fungi and phytochemicals in the dominant mangroves (Rhizophora mangle, Lacugularia racemosa, and Avicennia africana) of the Iko River Estuary in Nigeria was investigated. Standard analytical procedures were employed to measure antioxidant levels, while endophytic fungi were isolated, morphologically characterized, and identified using culture-dependent methods. The study identified various bioactive compounds, including alkaloids, saponins, tannins/phenols, flavonoids, deoxy-sugars, cardiac glycosides, and steroids, with their concentrations varying depending on the mangrove species and plant parts. Alkaloids were present in all mangrove samples, with particularly high concentrations in the leaves of white (5%) and red (4.2%) mangroves. Principal Component Analysis (PCA) was used to analyze the interaction between the endophytic fungi and phytochemicals, revealing a distinct affinity of certain fungal species for specific antioxidants in the mangroves. Fungal species such as Phoma sp., Rhizopus arrhizus, Aspergillus niger, and others exhibited a negative relationship with tannins, flavonoids, deoxysugars, and cardiac glycosides, but a positive affinity for saponins, alkaloids, and steroids. Conversely, Candida pseudotropicalis, Fusarium oxysporum, P. italicum, and others showed a positive affinity for tannins, flavonoids, cardiac glycosides, and deoxysugars, but a negative affinity for alkaloids, saponins, and steroids. Notably, Absidia sp. was the only frequently isolated species with a strong correlation (r value ≥75 at a 95% confidence limit) with deoxy-sugars, while Aspergillus niger, Aspergillus terreus, and Absidia sp. demonstrated significant relationships with alkaloids, saponins, and steroids. These phytochemicals, which serve as precursors to antioxidants with promising biotechnological applications, likely play a crucial role in determining the association of endophytic fungi with deltaic mangroves in Nigeria.

Biochemical characterization of a recombinant laccase from Halalkalibacterium halodurans C-125 and its application in the biotransformation of organic compounds.

This study aimed to produce an engineered recombinant laccase from extremophilic Halalkalibacterium halodurans C-125 (Lac-HhC-125) with higher protein yield, into a more active conformation and with properties that meet the fundamental needs of biotechnological application. The rLac-HhC125 was partially purified by size exclusion chromatography and concentrated by ultrafiltration (10kDa) with a yield of 57.6%. Oxidation reactions showed that adding 2mM CuSO4 to the assay solution led to activating the laccase. To increase its initial activity, the rLac-HhC125 was treated at 50°C for 20min before the assays, improving its performance by fourfold using the syringaldazine as a substrate. When treated with EDTA, methanol, ethanol, and DMSO, the rLac-HhC125 maintained more than 80% of its original activity. Interestingly, the acetonitrile induced a twofold activity of the rLac-HhC125. The putative rLac-HhC125 demonstrated a capability of efficient transformation of different organic compounds at pH 6, known as dye precursors, into coloured molecules. The rLac-HhC125 was active at high temperatures and alkaline pH, exhibited tolerance to organic solvents, and efficiently transformed different hydroxy derivatives into coloured compounds, which indicates that it can be used in various biotechnological processes.

Halophilic Pectinase-Producing Bacteria from Arthrocnemum macrostachyum Rhizosphere: Potential for Fruit–Vegetable Juice Processing

This study aimed to isolate salt-tolerant pectinolytic bacteria from the rhizosphere of a salt marsh plant and utilize their pectinases for the clarification of detox juice preparation. Sixteen halophilic bacterial strains were isolated from the rhizospheric soil of Arthrocnemum macrostachyum. The isolates were screened for pectinase activity, and two strains, ASA21 and ASA29, exhibited the highest pectinase production in the presence of 2.5% NaCl, reaching 13.3 and 14.1 IU mL−1, respectively. The strains were identified as Bacillus paralicheniformis and Paenibacillus sp. by 16S rDNA sequencing and phylogenetic analysis. Growth kinetics and pectinase production studies revealed that both strains produced pectinase during the log phase, with ASA29 demonstrating higher growth and pectinase titers. The pectinase from ASA29 exhibited enhanced activity in the presence of 3% NaCl. The pectinases from both strains were applied for the clarification of detox juice prepared from beetroot, carrots, and apples. The use of 20 IU mL−1 pectinase from ASA29 for 2–3 h yielded > 96% juice with high total phenolic content and antioxidant activities. This study highlights the potential of salt-tolerant pectinolytic bacteria from the rhizosphere for biotechnological applications, particularly in the clarification of juices with high salt content.

Mixed and membrane-separated culturing of synthetic cyanobacteria-yeast consortia reveals metabolic cross-talk mimicking natural cyanolichens

Metabolite exchange mediates crucial interactions in microbial communities, significantly impacting global carbon and nitrogen cycling. Understanding these chemically-mediated interactions is essential for elucidating natural community functions and developing engineered synthetic communities. This study investigated membrane-separated bioreactors (mBRs) as a novel tool to identify transient metabolites and their producers/consumers in mixed microbial communities. We compared three co-culture methods (direct mixed, 2-chamber mBR, and 3-chamber mBR) to grow a synthetic binary community of the cyanobacterium Synechococcus elongatus PCC 7942 and the fungus Rhodotorula toruloides NBRC 0880, as well as axenic S. elongatus. Despite not being natural lichen constituents, these organisms exhibited interactions resembling those in cyanolichens. S. elongatus fixed CO2 into sugars as the primary shared metabolite, while R. toruloides secreted various biochemicals, predominantly sugar alcohols, mirroring the metabolite exchange observed in natural lichens. The mBR systems successfully captured metabolite gradients and revealed rapidly consumed compounds, including TCA cycle intermediates and amino acids. Our approach demonstrated that the 2-chamber mBR optimally balanced metabolite exchange and growth dynamics. This study provides insights into cross-species metabolic interactions and presents a valuable tool for investigating and engineering synthetic microbial communities with potential applications in biotechnology and environmental science.

Toxigenic effects of sponges and benthic diatoms on marine invertebrates and their possible biotechnological applications

Secondary metabolites play important physiological roles being bioactive as defences against other organisms, or attractive signals used for various purposes, including reproduction. Their production and the emission in the environment may be viewed as an adaptive feature subjected to evolutionary selection. They were demonstrated to be useful for applications in various biotechnological fields, such as pharmaceutical, nutraceutical and cosmeceutical. Sponges and microalgae, including diatoms, are the most promising sources of bioactive compounds from the sea. We aimed at detecting the ecotoxicological effects of crude extracts and fractions obtained from three marine sponges, Geodia cydonium, Haliclona (Halichoclona) vansoesti and Agelas oroides and two benthic diatoms, Nanofrustulum shiloi and Cylindrotheca closterium on model marine organisms. We tested their effects on the Mediterranean purple sea urchin, Paracentrotus lividus, and on two diatoms, Phaeodactylum tricornutum and Cylindrotheca closterium, chosen because they are considered standard indicators for assessment of ecological impacts. Our results showed that extracts and fractions from both sponges and diatoms may be harmful for model invertebrates. However, eggs appeared “protected” from sponge allelochemicals when still unfertilized. The majority of sponge fractions exhibited noticeable impacts during the post-fertilization treatments. In contrast, fractions from diatoms notably increased the rate of malformations compared to the control, both in pre- and post-fertilization treatments.

High-Density Fermentation of Lactobacillus plantarum P6: Enhancing Cell Viability via Sodium Alginate Enrichment

Lactobacillus plantarum exhibits a wide range of beneficial physiological functions, including maintaining intestinal microbiota balance, reducing serum cholesterol, and promoting digestive health. According to the specific nutrient requirements of Lactobacillus plantarum P6, we investigated the effects of various carbon sources, nitrogen sources, trace elements, growth-promoting substances, as well as the initial pH and inoculum size on the growth of Lactobacillus plantarum P6 under fermentation conditions. The optimal growth conditions for Lactobacillus plantarum P6 were identified to facilitate high-density fermentation in small-scale fermenter production, achieving a cell concentration of 1.03 × 1011 CFU/mL. This resulted in a 2.5-fold increase in bacterial wet weight, and fermentation time was reduced to 12 h when utilizing a specific medium enriched with 0.2% sodium alginate. It is hypothesized that sodium alginate forms a protective film around the bacterial cells, promoting cell aggregation and enhancing self-coalescence, potentially triggering a bacterial community effect. These results provide a basis for the industrial-scale high-density cultivation of Lactobacillus plantarum, offering potential for enhanced biotechnological applications.

Machine-guided design of cell-type-targeting cis-regulatory elements.

Cis-regulatory elements (CREs) control gene expression, orchestrating tissue identity, developmental timing and stimulus responses, which collectively define the thousands of unique cell types in the body1-3. While there is great potential for strategically incorporating CREs in therapeutic or biotechnology applications that require tissue specificity, there is no guarantee that an optimal CRE for these intended purposes has arisen naturally. Here we present a platform to engineer and validate synthetic CREs capable of driving gene expression with programmed cell-type specificity. We take advantage of innovations in deep neural network modelling of CRE activity across three cell types, efficient in silico optimization and massively parallel reporter assays to design and empirically test thousands of CREs4-8. Through large-scale in vitro validation, we show that synthetic sequences are more effective at driving cell-type-specific expression in three cell lines compared with natural sequences from the human genome and achieve specificity in analogous tissues when tested in vivo. Synthetic sequences exhibit distinct motif vocabulary associated with activity in the on-target cell type and a simultaneous reduction in the activity of off-target cells. Together, we provide a generalizable framework to prospectively engineer CREs from massively parallel reporter assay models and demonstrate the required literacy to write fit-for-purpose regulatory code.

Reductive dehalogenase of Dehalococcoides mccartyi strain CBDB1 reduces cobalt- containing metal complexes enabling anodic respiration

Microorganisms capable of direct or mediated extracellular electron transfer (EET) have garnered significant attention for their various biotechnological applications, such as bioremediation, metal recovery, wastewater treatment, energy generation in microbial fuel cells, and microbial or enzymatic electrosynthesis. One microorganism of particular interest is the organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1, known for its ability to reductively dehalogenate toxic and persistent halogenated organic compounds through organohalide respiration (OHR), using halogenated organics as terminal electron acceptors. A membrane-bound OHR protein complex couples electron transfer to proton translocation across the membrane, generating a proton motive force, which enables metabolism and proliferation. In this study we show that the halogenated compounds can be replaced with redox mediators that can putatively shuttle electrons between the OHR complex and the anode, coupling D. mccartyi cells to an electrode via mediated EET. We identified cobalt-containing metal complexes, referred to as cobalt chelates, as promising mediators using a photometric high throughput methyl viologen-based enzyme activity assay. Through various biochemical approaches, we show that cobalt chelates are specifically reduced by CBDB1 cells, putatively by the reductive dehalogenase subunit (RdhA) of the OHR complex. Using cyclic voltammetry, we also demonstrate that cobalt chelates exchange electrons with a gold electrode, making them promising candidates for bioelectrochemical cultivation. Furthermore, using the AlphaFold 2-calculated RdhA structure and molecular docking, we found that one of the identified cobalt chelates exhibits favorable binding to RdhA, with a binding energy of approximately −28 kJ mol−1. Taken together, our results indicate that bioelectrochemical cultivation of D. mccartyi with cobalt chelates as anode mediators, instead of toxic halogenated compounds, is feasible, which opens new perspectives for bioremediation and other biotechnological applications of strain CBDB1.

Characterization of the Coriolopsis gallica DyP for Its Potential to Biotransform Various Fluoroquinolones

Coriolopsis gallica (Cga) is a white-rot fungus renowned for its ability to secrete ligninolytic enzymes that are capable of oxidizing phenolic compounds. This study aimed to investigate the biochemical characteristics of a dye-decolorizing peroxidase named CgaDyP1 and test its ability to biotransform antibiotics. CgaDyP1 was cloned and heterologously expressed in Escherichia coli. We fully characterized the biochemical properties of CgaDyP1 and evaluated its dye-decolorizing potential to confirm that it belongs to the DyP class of enzymes. We also tested its fluoroquinolone antibiotic biotransformation potential for possible biotechnological applications. Alignment of the primary amino acid sequence with DyP homolog sequences showed that CgaDyP1 has high similarity with other fungal DyPs. The recombinant CgaDyP1 exhibited activity on substrates such as ABTS and 2,6-dimethoxyphenol (DMP) with optimal performance at a pH of 3, although activity at pH 2.5, pH 4, and pH5 diminished over time. Thermostability tests indicated that the enzyme remains stable at temperatures between 30 °C and 50 °C and retains 70% of its initial activity after 180 min at 50 °C. Tests on the effect of hydrogen peroxide on CgaDyP1 activity found peak activity at 0.25 mM H2O2. CgaDyP1 decolorized five industrial dyes, and kinetics data confirmed that it belongs to the DyP class of enzymes. CgaDyP1 was shown to biotransform some of the 7 recalcitrant fluoroquinolone antibiotics tested here, including levofloxacin, moxifloxacin, and norfloxacin, and thus holds potential for biotechnological applications.

CRISPR-Cas systems in enterococci.

Enterococci are members of the microbiota of humans and other animals. They can also be found in the environment, associated with food, healthcare infections, and hospital settings. Due to their wide distribution, they are inserted in the One Health context. The selective pressure caused by the extensive use of antimicrobial agents in humans, animals, and agriculture has increased the frequency of resistance to various drugs among enterococcal species. CRISPR-Cas system, an important prokaryotic defense mechanism against the entry of mobile genetic elements, may prevent the acquisition of genes involved in antimicrobial resistance and virulence. This system has been increasingly used as a gene editing tool, which can be used as a way to recognize and inactivate genes of interest. Here, we conduct a review on CRISPR systems found in enterococci, considering their occurrence, structure and organization, mechanisms of action and use as a genetic engineering technology. Type II-A CRISPR-Cas systems were shown to be the most frequent among enterococcal species, and the orphan CRISPR2 was the most commonly found system (54.1%) among enterococcal species, especially in Enterococcus faecalis. Distribution of CRISPR systems varied among species. CRISPR systems had 1 to 20 spacers, with size between 23 and 37bp and direct repeat sequences from 25 to 37bp. Several applications of the CRISPR-Cas biotechnology have been described in enterococci, mostly in vitro, using this editing tool to target resistance- and virulence-related genes.

Molecular characterization, carbohydrate metabolism and tolerance to abiotic stress of Eremothecium coryli endophytic isolates from fruits of Momordica indica.

Yeasts are unicellular fungi that occur in a wide range of ecological niches, where they perform numerous functions. Furthermore, these microorganisms are used in industrial processes, food production, and bioremediation. Understanding the physiological and adaptive characteristics of yeasts is of great importance from ecological, biotechnological, and industrial perspectives. In this context, we evaluated the abilities to assimilate and ferment different carbon sources, to produce extracellular hydrolytic enzymes, and to tolerate salt stress, heavy metal stress, and UV-C radiation of two isolates of Eremothecium coryli, isolated from Momordica indica fruits. The two isolates were molecularly identified based on sequencing of the 18S-ITS1-5.8S-ITS2 region. Our isolates were able to assimilate nine carbon sources (dextrose, galactose, mannose, cellobiose, lactose, maltose, sucrose, melezitose, and pectin) and ferment three (glucose, maltose, and sucrose). The highest values of cellular dry weight were observed in the sugars maltose, sucrose, and melezitose. We observed the presence of hyphae and pseudohyphae in all assimilated carbon sources. The two isolates were also capable of producing amylase, catalase, pectinase, and proteases, with the highest values of enzymatic activity found in amylase. Furthermore, the two isolates were able to grow in media supplemented with copper, iron, manganese, nickel, and zinc and to tolerate saline stress in media supplemented with 5% NaCl. However, we observed a decrease in CFU at higher concentrations of these metals and NaCl. We also observed morphological changes in the presence of metals, which include changes in cell shape and cellular dimorphisms. The isolates were sensitive to UV-C radiation in the shortest exposure time (1min). Our findings reinforce the importance of endophytic yeasts for biotechnological and industrial applications and also help to understand how these microorganisms respond to environmental variations caused by human activities.

Development of a xylose-inducible and glucose-insensitive expression system for Parageobacillus thermoglucosidasius

Inducible expression systems are pivotal for governing gene expression in strain engineering and synthetic biotechnological applications. Therefore, a critical need persists for the development of versatile and efficient inducible expression mechanisms. In this study, the xylose-responsive promoter xylA5p and its transcriptional regulator XylR were identified in Parageobacillus thermoglucosidasius DSM 2542. By combining promoter xylA5p with its regulator XylR, fine-tuning the expression strength of XylR, and reducing the glucose catabolite repression on xylose uptake, we successfully devised a xylose-inducible and glucose-insensitive expression system, denoted as IExyl*. This system exhibited diverse promoter strengths upon induction with xylose at varying concentrations and remained unhindered in the presence of glucose. Moreover, we showed the applicability of IExyl* in P. thermoglucosidasius by redirecting metabolic flux towards riboflavin biosynthesis, culminating in a 2.8-fold increase in riboflavin production compared to that of the starting strain. This glucose-insensitive and xylose-responsive expression system provides valuable tools for designing optimized biosynthetic pathways for high-value products and facilitates future synthetic biology investigations in Parageobacillus.Key points• A xylose-inducible and glucose-insensitive expression system IExyl* was developed.• IExyl* was applied to enhance the riboflavin production in P. thermoglucosidasius• A tool for metabolic engineering and synthetic biology research in Parageobacillus strains.

Degradation of High Concentrations of Anthracene Using White-Rot Wood-Inhabiting Fungi and Investigation of Enzyme Activities

Owing to the production of lignin-modifying enzymes (LMEs), white-rot fungi (WRF) such as polypores are potent organisms in the biodegradation of xenobiotic pollutants. The nonspecific function of LMEs including laccase and manganese peroxidase (MnP), has enabled the use of WRF in biotechnological applications, particularly in bioremediation. In this study, 12 strains from nine white-rot basidiomycete genera viz., Ganoderma, Inocutis, Irpex, Lentinus, Lenzites, Oxyporus, Peniophora, Sanghuangporus, and Trametes were isolated from Iran and identified using morphological and molecular tools. The enzyme activity of laccase and manganese peroxidase that directly correlated with the biodegradation were determined, and the strains with the highest enzyme activities were evaluated for their ability to degrade 400 mg/L of anthracene over 28 days. Gas chromatography with flame ionization detector (GC-FID) revealed that four polypores viz., Trametes versicolor v21te, T. versicolor v22da, T. hirsuta, and Oxyporus sp. degraded 64%, 52%, 34%, and 20% of the anthracene, respectively. According to our analysis, the examined indigenous WRF are potentially useful candidates for the development of new mycoremediation techniques to degrade polycyclic aromatic hydrocarbons (PAHs).

Bioinformatic insights into sugar signaling pathways in sugarcane growth

The SnRK1, hexokinase, and TORC1 (TOR, LST8, RAPTOR) are three pivotal kinases at the core of sugar level sensing, significantly impacting plant metabolism and development. We retrieved and analyzed protein sequences of these three kinase pathways from seven sugarcane transcriptome and genome datasets, identifying protein domains, phylogenetic relationships, sequence ancestry, and in silico expression levels. Additionally, we predicted HXK subcellular localization and assessed its enzymatic activity in sugarcane leaves and culms along development in the field. We retrieved 11 TOR, 23 RAPTOR, 55 LST8, 95 SnRK1α, 98 HXK, and 14 HXK-like putative full-length sequences containing all the conserved domains. Most of these transcripts seem to share a common origin with the three ancestral species of sugarcane: Saccharum officinarum, Saccharum spontaneum, and Saccharum barberi. We accessed the expression profile of sequences from one sugarcane transcriptome. We found the highest enzymatic activity of HXK in culms in the first month, which, at this stage, provides carbon (sucrose) and nitrogen (amino acids) for initial plant development. Our approach places novel sugar sensing sequences that work as a guideline for further research into the underlying signaling mechanisms and biotechnology applications in sugarcane.

Systematic discovery of antibacterial and antifungal bacterial toxins.

Microorganisms use toxins to kill competing microorganisms or eukaryotic cells. Polymorphic toxins are proteins that encode carboxy-terminal toxin domains. Here we developed a computational approach to identify previously undiscovered, conserved toxin domains of polymorphic toxins within 105,438 microbial genomes. We validated nine short toxins, showing that they cause cell death upon heterologous expression in either Escherichia coli or Saccharomyces cerevisiae. Five cognate immunity genes that neutralize the toxins were also discovered. The toxins are encoded by 2.2% of sequenced bacteria. A subset of the toxins exhibited potent antifungal activity against various pathogenic fungi but not against two invertebrate model organisms or macrophages. Experimental validation suggested that these toxins probably target the cell membrane or DNA or inhibit cell division. Further characterization and structural analysis of two toxin-immunity protein complexes confirmed DNase activity. These findings expand our knowledge of microbial toxins involved in inter-microbial competition that may have the potential for clinical and biotechnological applications.

Laccase: A Green Biocatalyst Offers Immense Potential for Food Industrial and Biotechnological Applications.

Laccase, a multipurpose biocatalyst, is widely distributed across all kingdoms of life and plays a key role in essential biological processes such as lignin synthesis, degradation, and pigment formation. These functions are critical for fungal growth, plant-pathogen interactions, and maintenance of soil health. Due to its broad substrate specificity, multifunctional nature, and environmentally friendly characteristics, laccase is widely employed as a catalyst in various green chemistry initiatives. With its ability to oxidize a diverse range of phenolic and nonphenolic compounds, laccase has also been found to be useful as a food additive and for assessing food quality parameters. Ongoing advancements in research and technology are continually expanding the recognition of laccase's potential to address global environmental, health, and energy challenges. This review aims to provide critical insights into the applications of laccases in the biotechnology and food industry.