Search Results (9)

Background/Objectives: Limosilactobacillus fermentum KUB-D18, a heterofermentative lactic acid bacterium with promising probiotic properties, is known for promoting gut health and nutrient absorption. Originally isolated from chicken intestines, this strain demonstrates versatile metabolic capabilities in diverse gastrointestinal environments. However, the metabolic functions and sugar transport-related genes remain largely unexplored. This study thus aimed to dissect metabolic functions and sugar transports of L. fermentum KUB-D18. Methods: Next-generation and third-generation sequencing techniques using integrative genomic platform towards transportome analysis were performed. Results: The complete genome, sized at 2.12 Mbps with a GC content of 51.36%, revealed 2079 protein-encoding genes, of which 1876 protein functions were annotated and identified in top categories involved in amino acids, nucleotide, energy, and carbohydrate transports and metabolisms. Comparative genes analysis identified 50 core and 12 strain-specific genes linked to probiotic properties, e.g., acid resistances and bile tolerances, antioxidant functions, or anti-inflammatory properties. Further, sugar transportome analysis uncovered 57 transporter genes, demonstrating diverse carbon utilization and phosphotransferase (PTS) systems, corroborated by API 50 CHL test results for carbohydrate metabolism profile. Conclusions: These findings enhance the comprehensive metabolic understanding of L. fermentum KUB-D18, supporting its industrial potential and applications in engineered probiotics. Full article

Escherichia coli is the best-known model for the biotechnological production of many biotechnological products, including housekeeping and heterologous primary and secondary metabolites and recombinant proteins, and is an efficient biofactory model to produce biofuels to nanomaterials. Glucose is the primary substrate used as the carbon source for laboratory and industrial cultivation of E. coli for production purposes. Efficient growth and associated production and yield of desired products depend on the efficient sugar transport capabilities, sugar catabolism through the central carbon catabolism, and the efficient carbon flux through specific biosynthetic pathways. The genome of E. coli MG1655 is 4,641,642 bp, corresponding to 4702 genes encoding 4328 proteins. The EcoCyc database describes 532 transport reactions, 480 transporters, and 97 proteins involved in sugar transport. Nevertheless, due to the high number of sugar transporters, E. coli uses preferentially few systems to grow in glucose as the sole carbon source. E. coli nonspecifically transports glucose from the extracellular medium into the periplasmic space through the outer membrane porins. Once in periplasmic space, glucose is transported into the cytoplasm by several systems, including the phosphoenolpyruvate-dependent phosphotransferase system (PTS), the ATP-dependent cassette (ABC) transporters, and the major facilitator (MFS) superfamily proton symporters. In this contribution, we review the structures and mechanisms of the E. coli central glucose transport systems, including the regulatory circuits recruiting the specific use of these transport systems under specific growing conditions. Finally, we describe several successful examples of transport engineering, including introducing heterologous and non-sugar transport systems for producing several valuable metabolites. Full article

A carbohydrate binding module 68 (CBM68) of pullulanase from Anoxybacillus sp. LM18-11 was used to enhance the secretory expression of a thermostable α-amylase (BLA702) in Bacillus subtilis, through an atypical secretion pathway. The extracellular activity of BLA702 guided by CBM68 was 1248 U/mL, which was 12.6 and 7.2 times higher than that of BLA702 guided by its original signal peptide and the endogenous signal peptide LipA, respectively. A single gene knockout strain library containing 51 genes encoding macromolecular transporters was constructed to detect the effect of each transporter on the secretory expression of CBM68-BLA702. The gene knockout strain 0127 increased the extracellular amylase activity by 2.5 times. On this basis, an engineered strain B. subtilis 0127 (AmyE::BLA702-NprB::CBM68-BLA702-PrsA) was constructed by integrating BLA702 and CBM68-BLA702 at the AmyE and NprB sites in the genome of B. subtilis 0127, respectively. The molecular chaperone PrsA was overexpressed, to reduce the inclusion body formation of the recombinant enzymes. The highest extracellular amylase activity produced by B. subtilis 0127 (AmyE::BLA702-NprB::CBM68-BLA702-PrsA) was 3745.7 U/mL, which was a little lower than that (3825.4 U/mL) of B. subtilis 0127 (pMAC68-BLA702), but showing a better stability of passage. This newly constructed strain has potential for the industrial production of BLA702. Full article

(Photo-)electrocatalytic artificial photosynthesis driven by electrical and/or solar energy that converts water (H₂O) and carbon dioxide (CO₂) into hydrogen (H₂), carbohydrates and oxygen (O₂), has proven to be a promising and effective route for producing clean alternatives to fossil fuels, as well as for storing intermittent renewable energy, and thus to solve the energy crisis and climate change issues that we are facing today. Basic (photo-)electrocatalysis consists of three main processes: (1) light absorption, (2) the separation and transport of photogenerated charge carriers, and (3) the transfer of photogenerated charge carriers at the interfaces. With further research, scientists have found that these three steps are significantly affected by surface and interface properties (e.g., defect, dangling bonds, adsorption/desorption, surface recombination, electric double layer (EDL), surface dipole). Therefore, the catalytic performance, which to a great extent is determined by the physicochemical properties of surfaces and interfaces between catalyst and reactant, can be changed dramatically under working conditions. Common approaches for investigating these phenomena include X-ray photoelectron spectroscopy (XPS), X-ray absorption spectroscopy (XAS), scanning probe microscopy (SPM), wide angle X-ray diffraction (WAXRD), auger electron spectroscopy (AES), transmission electron microscope (TEM), etc. Generally, these techniques can only be applied under ex situ conditions and cannot fully recover the changes of catalysts in real chemical reactions. How to identify and track alterations of the catalysts, and thus provide further insight into the complex mechanisms behind them, has become a major research topic in this field. The application of in situ/operando characterization techniques enables real-time monitoring and analysis of dynamic changes. Therefore, researchers can obtain physical and/or chemical information during the reaction (e.g., morphology, chemical bonding, valence state, photocurrent distribution, surface potential variation, surface reconstruction), or even by the combination of these techniques as a suite (e.g., atomic force microscopy-based infrared spectroscopy (AFM-IR), or near-ambient-pressure STM/XPS combined system (NAP STM-XPS)) to correlate the various properties simultaneously, so as to further reveal the reaction mechanisms. In this review, we briefly describe the working principles of in situ/operando surface/interface characterization technologies (i.e., SPM and X-ray spectroscopy) and discuss the recent progress in monitoring relevant surface/interface changes during water splitting and CO₂ reduction reactions (CO₂RR). We hope that this review will provide our readers with some ideas and guidance about how these in situ/operando characterization techniques can help us investigate the changes in catalyst surfaces/interfaces, and further promote the development of (photo-)electrocatalytic surface and interface engineering. Full article

Aspergillus niger is an important industrial workhorse for the biomanufacturing of organic acids, proteins, etc. Well-controlled genetic regulatory elements, including promoters, are vital for strain engineering, but available strong promoters for A. niger are limited. Herein, to efficiently assess promoters, we developed an accurate and intuitive fluorescent-auxotrophic selection workflow based on mCherry, pyrG, CRISPR/Cas9 system, and flow cytometry. With this workflow, we characterized six endogenous constitutive promoters in A. niger. The endogenous glyceraldehyde-3-phosphate dehydrogenase promoter PgpdAg showed a 2.28-fold increase in promoter activity compared with the most frequently used strong promoter PgpdAd from A. nidulans. Six predicted conserved motifs, including the gpdA-box, were verified to be essential for the PgpdAg activity. To demonstrate its application, the promoter PgpdAg was used for enhancing the expression of citrate exporter cexA in a citric acid-producing isolate D353.8. Compared with the cexA controlled by PgpdAd, the transcription level of the cexA gene driven by PgpdAg increased by 2.19-fold, which is consistent with the promoter activity assessment. Moreover, following cexA overexpression, several genes involved in carbohydrate transport and metabolism were synergically upregulated, resulting in up to a 2.48-fold increase in citric acid titer compared with that of the parent strain. This study provides an intuitive workflow to speed up the quantitative evaluation of A. niger promoters and strong constitutive promoters for fungal cell factory construction and strain engineering. Full article

The quality of Lily cut flower was determined by the quality of bulbs. During the process of vernalization and flower bud differentiation, sugar massively accumulated in the bulb, which influenced the bulb development. However, the details of sugar genes’ regulation mechanism for these processes were not fully understood. Here, morphological physiology, transcriptomes and gene engineering technology were used to explore this physiological change. Seventy-two genes of 25 kinds of sugar metabolism-related genes were annotated after re-analyzing transcriptome data of Oriental hybrid lily ‘Sorbonne’ bulbs, which were generated on Hiseq Illumina 2000. The results showed that these genes were closely related to lily bulb vernalization and development. Combining gene expression pattern with gene co-expression network, five genes (Contig5669, Contig13319, Contig7715, Contig1420 and Contig87292) were considered to be the most potential signals, and the sucrose transporter gene (SUT) was the focus of this study. Carbohydrate transport pathway and genes’ regulation mechanism were inferred through a physiological and molecular test. SUT seemed to be the sugar sensor that could sense and regulate sugar concentration, which might have effects on other genes, such as FT, LFY and so on. LoSUT2 and LoSUT4 genes were cloned from Oriental hybrid lily ‘Sorbonne’ by RACE, which was the first time for these genes in Oriental hybrid lily ‘Sorbonne’. The physiological properties of these proteins were analyzed such as hydrophobicity and phosphorylation. In addition, secondary and tertiary structures of proteins were predicted, which indicated the two proteins were membrane proteins. Their cellular locations were verified through positioning the experiment of the fluorescent vector. They were highly expressed in cells around phloem, which illustrated the key role of these genes in sugar transport. Furthermore, transient expression assays showed that overexpressed LoSUT2 and LoSUT4 in Arabidopsis thaliana bloomed significantly earlier than the wild type and the expression of FT, SOC1 and LFY were also affected by LoSUT2 and LoSUT4, which indicated that LoSUT2 and LoSUT4 may regulate plants flowering time. Full article

Currently, the fermentation technology for recycling agriculture waste for generation of alternative renewable biofuels is getting more and more attention because of the environmental merits of biofuels for decreasing the rapid rise of greenhouse gas effects compared to petrochemical, keeping in mind the increase of petrol cost and the exhaustion of limited petroleum resources. One of widely used biofuels is bioethanol, and the use of yeasts for commercial fermentation of cellulosic and hemicellulosic agricultural biomasses is one of the growing biotechnological trends for bioethanol production. Effective fermentation and assimilation of xylose, the major pentose sugar element of plant cell walls and the second most abundant carbohydrate, is a bottleneck step towards a robust biofuel production from agricultural waste materials. Hence, several attempts were implemented to engineer the conventional Saccharomyces cerevisiae yeast to transport and ferment xylose because naturally it does not use xylose, using genetic materials of Pichia stipitis, the pioneer native xylose fermenting yeast. Recently, the nonconventional yeast Spathaspora passalidarum appeared as a founder member of a new small group of yeasts that, like Pichia stipitis, can utilize and ferment xylose. Therefore, the understanding of the molecular mechanisms regulating the xylose assimilation in such pentose fermenting yeasts will enable us to eliminate the obstacles in the biofuels pipeline, and to develop industrial strains by means of genetic engineering to increase the availability of renewable biofuel products from agricultural biomass. In this review, we will highlight the recent advances in the field of native xylose metabolizing yeasts, with special emphasis on S. passalidarum for improving bioethanol production. Full article

Iron plays an essential role in all organisms and is involved in the structure of many biomolecules. It also regulates the Fenton reaction where highly reactive hydroxyl radicals occur. Iron is also important for microbial biodiversity, health and nutrition. Excessive iron levels can cause oxidative damage in cells. Saccharomyces cerevisiae evolved mechanisms to regulate its iron levels. To study the iron stress resistance in S. cerevisiae, evolutionary engineering was employed. The evolved iron stress-resistant mutant “M8FE” was analysed physiologically, transcriptomically and by whole genome re-sequencing. M8FE showed cross-resistance to other transition metals: cobalt, chromium and nickel and seemed to cope with the iron stress by both avoidance and sequestration strategies. PHO84, encoding the high-affinity phosphate transporter, was the most down-regulated gene in the mutant, and may be crucial in iron-resistance. M8FE had upregulated many oxidative stress response, reserve carbohydrate metabolism and mitophagy genes, while ribosome biogenesis genes were downregulated. As a possible result of the induced oxidative stress response genes, lower intracellular oxidation levels were observed. M8FE also had high trehalose and glycerol production levels. Genome re-sequencing analyses revealed several mutations associated with diverse cellular and metabolic processes, like cell division, phosphate-mediated signalling, cell wall integrity and multidrug transporters. Full article

Extracellular vesicles (EVs) are membrane enclosed micro- and nano-sized vesicles that are secreted from almost every species, ranging from prokaryotes to eukaryotes, and from almost every cell type studied so far. EVs contain repertoire of bioactive molecules such as proteins (including enzymes and transcriptional factors), lipids, carbohydrates and nucleic acids including DNA, coding and non-coding RNAs. The secreted EVs are taken up by neighboring cells where they release their content in recipient cells, or can sail through body fluids to reach distant organs. Since EVs transport bioactive cargo between cells, they have emerged as novel mediators of extra- and intercellular activities in local microenvironment and inter-organ communications distantly. Herein, we review the activities of EV-associated matrix-remodeling enzymes such as matrix metalloproteinases, heparanases, hyaluronidases, aggrecanases, and their regulators such as extracellular matrix metalloproteinase inducers and tissue inhibitors of metalloproteinases as novel means of matrix remodeling in physiological and pathological conditions. We discuss how such EVs act as novel mediators of extracellular matrix degradation to prepare a permissive environment for various pathological conditions such as cancer, cardiovascular diseases, arthritis and metabolic diseases. Additionally, the roles of EV-mediated matrix remodeling in tissue repair and their potential applications as organ therapies have been reviewed. Collectively, this knowledge could benefit the development of new approaches for tissue engineering. Full article