javascript:void(0) Antioxidants are known for their health beneficial effects by reducing oxidative damage, increasing free radical scavenging rate, blocking and terminating oxidation reactions. They have been widely used as supplements in food, cosmetics, nutraceuticals and medicines. Emerging researches increasingly indicate that the development of diverse research methodologies and the exploration of new microorganisms for heterogeneous synthesis of antioxidants play an indispensable role in socioeconomic development. In this study, we engineered Saccharomyces boulardii for probiotic supplementation of antioxidants such as L-ergothioneine (EGT). We first constructed double knockout of ura3 and trp1 gene in S. boulardii, to facilitate plasmid-based expressions. To further enable effective genome editing of S. boulardii, we implemented the PiggyBac system to transpose the heterologous gene expression cassettes into the chromosomes of S. boulardii. By using enhanced green fluorescent protein (EGFP) as the reporter gene, we achieved random chromosomal integration of EGFP expression cassette. EGT-producing strains were subsequently obtained via the PiggyBac transposon system. One isolated S. boulardii mutant produced EGT at 17.50 mg/L after 120 h cultivation. In summary, we have applied the PiggyBac transposon system to S. boulardii for the first time for genetic engineering. The engineered probiotic yeast S. boulardii has been endowed with new antioxidant properties and produces EGT. It has potential applications in developing novel therapeutics and dietary supplements for the prevention and treatment of gastrointestinal disorders.

Mesenchymal stem cells (MSCs) derived exosomes, as a cell-free therapy to replace MSCs, have higher safety and great potential in regenerative medicine. The isolation of exosomes is a challenge that complicates their application. The commonly used ultracentrifugation and tangential flow filtration methods are inconvenient due to the need for expensive instruments and ultrafilter membranes. The cost of commercial kits limits their application in handling large samples. PEG precipitation is convenient and cost-effective. However, there is a need for optimized and standardized isolation methods based on PEG precipitation in different cell types or liquids to ensure consistent quality and yield. In this work, we optimized the PEG precipitation method for exosomes isolation and compared its effectiveness to two commonly used methods: ultracentrifugation (UC) and commercial exosome isolation kits (ExoQuick). The recovery rate of the optimized PEG method ( about 61.74%) was comparable to that of the commercial ExoQuick kit (about 62.19%), which was significantly higher than UC (about 45.80%). Exosome cargo analysis validated no significant differences in miRNA and protein profiles associated with the proliferation and migration of exosomes isolated by UC and PEG precipitation, which was confirmed by gap closure and CCK8 assays. These findings suggest that PEG-based exosomes isolation could be a highly efficient and high-quality method and may facilitate the development of exosome-based therapies for regenerative medicine.

After the COVID-19 pandemic, several companies and organisations are actively developing, scaling up and optimising mRNA manufacturing processes to produce their vaccines and therapeutics. Herein, we evaluated tangential flow filtration (TFF) for high-recovery, and high-purity separation of mRNA from unreacted NTPs in the IVT reaction mixture. For the first time, the membrane fouling behaviour caused by mRNA and separation of nucleoside triphosphates (NTPs) was mathematically modelled. The mRNA membrane fouling model is necessary for optimising mRNA separation processes, designing a suitable strategy for membrane clean-up, estimating the end of production life and reducing the process cost. Recovery greater than 70% mRNA without degradation was obtained by implementing a capacity load of ~19 g/m2, <2.5 psi transmembrane pressure (TMP) and feed flux of 300 LMH. This approach enables the purification of multiple RNA drug substance sequences for the prevention and treatment of a wide range of diseases.

Squalene (C30H50) is an acyclic triterpenoid compound renowned for its myriad physiological functions, such as anticancer and antioxidative properties, rendering it invaluable in both the food and pharmaceutical sectors. Owing to the constraints on natural resources, microbial fermentation has emerged as a prominent trend. Schizochytrium sp. known to harbor the intact Mevalonate (MVA) pathway, possesses the inherent capability to biosynthesize squalene. However, there is a dearth of reported key genes in both the MVA pathway and the squalene synthesis pathway, along with the associated promoter elements for their modification. This study commenced by cloning and characterizing 13 endogenous promoters derived from transcriptome sequencing data. Subsequently, five promoters exhibiting varying expression intensities were chosen from the aforementioned pool to facilitate the overexpression of the squalene synthase gene SQS, pivotal in the MVA pathway. Ultimately, a transformed strain designated as SQS-3626, exhibiting squalene production 2.8 times greater than that of the wild-type strain, was identified. Lastly, the optimization of nitrogen source concentrations and trace element contents in the fermentation medium was conducted. Following 120 hours of fed-batch fermentation, the accumulated final squalene yield in the transformed strain SQS-3626 reached 2.2 g/L.

Plant gene editing technology has significantly advanced in recent years, thereby transforming both biotechnological research and agricultural practices. This review provides a comprehensive summary of recent advancements in this rapidly evolving field, showcasing significant discoveries from improved transformation efficiency to advanced construct design. The primary focus is on the maturation of the CRISPR-Cas9 system, which has emerged as a powerful tool for precise gene editing in plants. Through a detailed exploration, we elucidate the intricacies of integrating genetic modifications into plant genomes, shedding light on transport mechanisms, transformation techniques, and optimization strategies specific to CRISPR constructs. Furthermore, we explore the initiatives aimed at extending the frontiers of gene editing to non-model plant species, showcasing the growing scope of this technology. Overall, this comprehensive review highlights the significant impact of recent advancements in plant gene editing, illuminating its transformative potential in driving agricultural innovation and biotechnological progress.

A new method has been developed to improve the detection of norovirus (NoV) in complex fecal samples using nanomaterials-based immunoassays. The method involves using multifunctional trimetallic nanoparticles, known as Ag@Fe3O4@Au NPs. These nanoparticles consist of a core of silver (Ag) and a shell of iron oxide (Fe3O4) decorated with isolated gold (Au) nanoparticles. The nanoparticles have enhanced catalytic activity, making them an ideal nanocatalyst for reducing substrate molecules. The developed Ag@Fe3O4@Au NPs-based immunoassay achieved a limit of detection (LOD) of 1.9 pg/mL for norovirus-like particles (NoV-LP) and 6.97 fecal NoV copy number/mL. Achieving a LOD under 10 copy numbers is important for detailed protein analysis. The study also found that heat treatment of fecal suspension at 65°C was necessary to prevent the degradation of the target analyte for sensitive NoV detection. This work successfully combined multifunctional nanocatalysts for advanced immunoassays, which could contribute to developing nano-biosensing platforms.

In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for rAAV production by targeting the STAT1 locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout success. Selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile and potency, among others, were assessed. Our study also established a comprehensive analytical workflow to detect and evaluate the gene knockouts generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.

Laccases have a wide range of uses in the textile industry for their ability to decolorize dyes, modify the surface of fabrics and bleach textiles. Screening a laccase with high thermal stability and alkali tolerance suitable for textile applications is challenging. In this study, we identified a novel alkaline laccase, LacCT, from Caldalkalibacillus thermarum, and expressed it in Escherichia coli. It showed an optimal temperature of 65°C and a pH range of high stability from 6.0 to 10.0, with an optimum pH of 7.5. Through rational design, the thermal stability of the best variant, G190P/Q254Y/G336M/D510F (LacCT-11), was significantly improved, exhibiting a half-life of 63.2 minutes at 60°C, 1.8 times longer than the wild type. This research presents a new laccase with significant potential for decolorizing textile wastewater and enhancing the ramie degumming process.

The Chinese hamster ovary (CHO) cell is a fibroblast-like cell that produces proteins with post-translational modifications similar to human glycosylation. It is widely used in the production of recombinant therapeutic proteins and monoclonal antibodies. Culturing CHO cells typically requires the addition of a certain proportion of fetal bovine serum (FBS) to maintain cell proliferation and passaging. However, serum is characterized by its complex composition, batch-to-batch variability, high cost, and potential risk of exogenous contaminants such as mycoplasma and viruses, which impact the purity and safety of the synthesized proteins. Therefore, search for serum alternatives and development of serum-free media for CHO-based protein biomanufacturing are of great significance. This review systematically summarizes the application advantages of CHO cells and strategies for high-density expression. It highlights the developmental trends of serum substitutes from human platelet lysates to animal-free extracts and microbial-derived substances and elucidates the mechanisms by which these substitutes enhance CHO cell culture performance and recombinant protein production, aiming to provide theoretical guidance for exploring novel serum alternatives and developing serum-free media for CHO cells.

The mutual interactions of ER resident proteins in the ER maintain its functions, prompting the protein folding, modification, and transportation. Here, a new method, named YST-PPI (YESS-based Split fast TEV protease System for Protein-protein Interaction) was developed, targeting the characterization of protein interactions in ER. YST-PPI method integrated the YESS system, split-TEV technology and endoplasmic reticulum retention signal peptide (ERS) to provide an effective strategy for studying ER in situ PPIs in a fast and quantitative manner. The interactions among 15 ER resident proteins of S. cerevisiae were explored using the YST-PPI system, and their interaction network map was constructed, in which more than 74 interacting resident protein pairs were identified. Our studies also showed that Lhs1p plays a critical role in regulating the interactions of most of the ER resident proteins, expect the Sil1p, indicating its potential role in controlling the ER molecular chaperones. Moreover, the mutual interaction revealed by our studies further confirmed that the ER resident proteins perform their functions in a synergetic way and multimer complex might be formed during the process.

The performance of industrial strains has gradually improved with the rapid development of synthetic biotechnology. The production efficiency of traditional batch and fed-batch culture is limited and product quality varies since both are dynamic processes, whereas multi-stage continuous culture can maximise the production efficiency of specific fermentation processes and achieve consistent product quality. However, each single-stage fermentation under multi-stage continuous fermentation requires accurate steady-state control, and a model with adequate accuracy is required for designing and controlling a multi-stage continuous fermentation process. At present, there are few reports on kinetic models for the control of multi-stage continuous fermentation. In this work, we constructed a hybrid model for Saccharomyces cerevisiae multi-stage continuous culture, taking both oxygen limitation and Crabtree effect. The accuracy of the model was ~ 80%, advantages and limitations of the model are discussed and potential improvement strategy is proposed.

DNA ligases catalyze bond formation in the backbone of nucleic acids via the formation of a phosphodiester bond between adjacent 5’ phosphates and 3’ hydroxyl groups on one strand of the duplex. While DNA ligases preferentially ligate single breaks in double stranded DNA (dsDNA), they are capable of ligating a multitude of other nucleic acids substrates like blunt-ended dsDNA, TA overhangs, short overhangs and various DNA-RNA hybrids. Here we report a novel DNA ligase from Chronobacter phage CR 9 (AZ R2D Ligase) with an unexpected DNA-to-RNA ligation activity. The R2D ligase shows excellent efficiency when ligating DNA to either end of RNA molecules using a DNA template. Furthermore, we show that DNA can be ligated simultaneously to both the 5’ and 3’ ends of microRNA-like molecules in a single reaction mixture. Abortive adenylated side product formation is suppressed at lower ATP concentrations and the ligase reaction reaches near completion when ligating RNA to DNA or DNA to RNA. The ligation of a DNA strand to the 5’-PO4 end of RNA is unique among the commercially available ligases and may facilitate novel workflows in microRNA analysis, RNA sequencing and in the preparation of chimeric guide DNA-RNA for gene editing applications.

Amino acids, including asparagine, aspartate, glutamine, and glutamate, play important roles in the purine and pyrimidine biosynthesis as well as serve as anaplerotic sources fueling the tricarboxylic acid (TCA) cycle for mitochondrial energy generation in mammalian cells. Despite extensive studies on glutamine and glutamate in CHO cell cultures, the roles of asparagine and aspartate, especially in feed media, remain underexplored. In this study, we utilized the CHO genome scale model to first deeply characterize the intracellular metabolic states of CHO cells cultured in different combinations of basal and feed media to understand the traits of asparagine/aspartate-dependent and glutamate-dependent feeds. Subsequently, we identified the critical role of asparagine and aspartate in the feed media as anaplerotic sources and conduct in silico simulations to ascertain their optimal ratios to improve cell culture performance. Finally, based on the model simulations, we reformulated the feed media by tailoring the concentrations of asparagine and aspartate. Our experimental data reveal a CHO cell preference for asparagine compared with aspartate, and thus maintaining an optimal ratio of these amino acids is a key factor for achieving optimal CHO cell culture performance in biopharmaceutical production.

Schizochytrium sp. is a heterotrophic microorganism capable of accumulating polyunsaturated fatty acids, and has achieved industrial production of docosahexaenoic acid (DHA). It also has the potential for eicosapentaenoic acid (EPA) production. In this study, it was found that the cell growth, lipid synthesis and fatty acid composition of Schizochytrium sp. were significantly affected by the level of cobalamin in the medium, especially with regards to the content of EPA in the fatty acids. The content of EPA in the fatty acids increased 17.91 times, reaching 12.0%, but cell growth and lipid synthesis were significantly inhibited under cobalamin deficiency. The response mechanism for this phenomenon was revealed through combined lipidomic and transcriptomic analysis. Although cell growth was inhibited under cobalamin deficiency, the genes encoding key enzymes in central carbon metabolism were still up-regulated to provide precursors (Acetyl-CoA) and reducing power (NADPH) for the synthesis and accumulation of fatty acids. Moreover, the main lipid subclasses observed during cobalamin deficiency were glycerolipids (including glycerophospholipids), with EPA primarily distributed in them. The genes involved in the biosynthesis of these lipid subclasses were significantly up-regulated, such as the key enzymes in the Kennedy pathway for the synthesis of triglycerides. Thus, this study provided insights into the specific response of Schizochytrium sp. to cobalamin deficiency and identified a subset of new genes that can be engineered for modification.

Laccases have shown to be efficient biocatalysts for the removal of recalcitrant pollutants from wastewater. Thus, they catalyse the oxidation of a wide variety of organic compounds by reducing molecular oxygen to water. However, the use of free laccases holds several drawbacks such as poor reusability, high cost, low stability and sensibility to different denaturing agents that may occur in wastewater. Such drawbacks can be circumvented by immobilising laccase enzymes in/on solid carriers. Hence, during the last decades different approaches considering various techniques and solid carriers to immobilise laccase enzymes have been developed and tested for the removal of pollutants from wastewater. To scale up wastewater treatment bioprocesses, the immobilised laccases are placed in different reactor configurations.

This study evaluated the performance of biofilm reactors inoculated with azo dye degrading Shewanella for the decolorization of Reactive Black 5 (RB5), using three different carrier materials, namely almond shell biochar, moving bed biofilm reactor (MBBR), and polypropylene carrier (PPC). The reactors were fed with low-nutrient artificial wastewater containing RB5 for a long term, and all three carriers showed good RB5 decolorization performance, with varying efficiencies. LC-MS analysis revealed significant differences in the degradation pathways of RB5 among the carriers, indicating the role of carrier materials and microbial communities. The MBBR carrier exhibited good stability due to its rough surface and microbial aggregates. Sequencing results revealed significant differences in the microbial community structure among the carriers. Shewanella was the dominant functional bacteria in the MBBR and PPC carriers, while highly efficient degrading microbial communities were observed on the biochar carrier. Overall, the physicochemical properties of the carrier materials had a significant impact on the microbial community in the reactor, which affected the degradation efficiency of RB5. These findings provide valuable insights into the optimization of biofilm reactors for dye-containing wastewater treatment.

The combination of single-cell RNA sequencing and microdissection techniques that preserves positional information has become a major tool for spatial transcriptome analyses. However, high costs and time requirements, especially for experiments at the single cell scale, make it challenging for this approach to meet the demand for increased throughput. Therefore, we proposed combinational DNA barcode (CDB)-seq as a medium-throughput, multiplexed approach combining Smart-3SEQ and CDB magnetic microbeads for transcriptome analyses of microdissected tissue samples. We conducted a comprehensive comparison of conditions for CDB microbead preparation and related factors and then applied CDB-seq to RNA extracts, fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) mouse brain tissue samples. CDB-seq transcriptomic profiles of tens of microdissected samples could be obtained in a simple, cost-effective way, providing a promising method for future spatial transcriptomics.

The isopentenol utilization pathway (IUP) is potential in terpenoids synthesis. This study aimed to construct IUP-employed E. coli chassis for stably synthesizing terpenoids. As to effectiveness, promotor engineering strategy was employed to regulate IUP expression level, while ribosome-binding site (RBS) library of the key enzyme was constructed for screening the optimal RBS, followed by optimization of concentration of inducer and substrates, the titer of reporting production, lycopene, from 0.087 to 8.67 mg/OD600. As about stability, the IUP expression cassette was integrated into the genome through transposition tool based on CRISPR-associated transposases. Results showed that the strain with 13 copies produced 1.78-fold lycopene titer that of the controlled strain with IUP-harbored plasmid, and it exhibited stable expression after ten successions while the plasmid loss was observed in the controlled strain in the 3rd succession. This strategy provides valuable information for rapid construction of highly effective and stable chassis employing IUP for terpenoids production.