Search Results (47)

Lipid nanodiscs are synthetic nanoparticles capable of solubilizing lipophilic drugs and have been shown to improve the potency of the antifungal Amphotericin B (AmphB) against various fungal pathogens. In this study, the SpyCatcher–SpyTag covalent labeling system was used to couple AmphB-loaded Apolipoprotein A1 (ApoA1) lipid nanodiscs to the receptor domain of Dectin-1, which binds to β-1,3/1,6 glucans present in many fungal cell walls. Denaturing protein gel electrophoresis demonstrated that ApoA1-SpyTag003 lipid nanodiscs could be covalently labeled with SpyCatcher003-Dectin-1-superfolder GFP (sfGFP). In microtiter growth assays with Saccharomyces cerevisiae, Dectin-1 AmphB nanodiscs displayed an IC₅₀ 1.5-fold lower than uncoupled AmphB nanodiscs and 2.8-fold lower than AmphB-only controls. Nanodiscs without AmphB and SpyCatcher003-Dectin-1-sfGFP themselves did not inhibit yeast growth. Fluorescence microscopy showed that SpyCatcher003-Dectin-1-sfGFP binds to yeast cell walls and accumulated at hot spots, matching the budding scar enrichment pattern previously described for other Dectin-1 fusion proteins. Together these results indicate that Dectin-1 fusions can target AmphB-loaded lipid nanodiscs to fungal cell walls and improve drug delivery. The results here establish the use of a modular SpyCatcher–SpyTag coupling system for targeting drug-loaded lipid nanodiscs to different cells or tissues, thereby increasing drug retention at infection sites, increasing drug potency, and reducing harmful side-effects. Full article

Respiratory syncytial virus (RSV) poses a significant global health challenge, particularly affecting infants, the elderly, and immunocompromised individuals. Despite recent progress in the development of vaccines and monoclonal antibodies, effective antiviral therapies remain limited. To advance the discovery of antiviral drugs, we have developed a dual-reporter RSV minigenome system, providing a safe and robust platform for antiviral evaluation. This system incorporates NanoLuc luciferase and superfolder GFP (sfGFP) linked by a self-cleaving P2A peptide, allowing for the simultaneous detection of orthogonal signals. Validation with L polymerase inhibitors confirmed the system’s reliability for screening small-molecule inhibitors. The linear correlation observed between the reporter signals enhances the assay’s reliability for antiviral assessment. This dual-reporter minigenome system advances targeted therapeutic strategies against RSV. Full article

Monoacylglycerols (MAGs) are significant intermediate byproducts in the hydrolysis of oils and fats. The accumulation of MAGs not only reduces the quality and purity of the final products in biodiesel production and edible oil refining but also poses challenges for downstream separation processes. Therefore, the development of efficient biocatalysts for the specific MAG conversion is of great industrial importance. The lipase from Aspergillus oryzae (AOL) has shown potential for lipid modification; however, the wild-type enzyme (WT) suffers from poor solubility, tendency to aggregate, and low specific activity towards MAGs in aqueous systems, which severely restricts its practical application. In this study, a combinatorial protein engineering strategy was employed to overcome these limitations. We integrated fusion protein technology with rational design to enhance both the functional expression and catalytic efficiency of AOL. Firstly, the superfolder green fluorescent protein (sfGFP) was fused to the N-terminus of AOL. The results indicated that the sfGFP fusion tag significantly improved the solubility and stability of the enzyme, preventing the formation of inclusion bodies. The fusion protein sfGFP-AOL exhibited a MAG conversion rate of approximately 65%, confirming the positive impact of the fusion tag on enzyme developability. To further boost catalytic performance, site-directed mutagenesis was performed based on structural analysis. Among the variants, the mutant sfGFP-Y92Q emerged as the most potent candidate. In the MAG conversion, sfGFP-Y92Q achieved a conversion rate of 98%, which was not only significantly higher than that of sfGFP-AOL but also outperformed the widely used commercial immobilized lipase, Novozym 435 (~54%). Structural modeling and docking analysis revealed that the Y92Q mutation optimized the geometry of the active site. The substitution of Tyrosine with Glutamine at position 92 likely enlarged the substrate-binding pocket and altered the local electrostatic environment, thereby relieving steric hindrance and facilitating the access of the bulky MAG substrate to the catalytic center. In conclusion, this work demonstrates that the synergistic application of sfGFP fusion and rational point mutation (Y92Q) can dramatically transform the catalytic properties of AOL. The engineered sfGFP-Y92Q variant serves as a robust and highly efficient biocatalyst for MAG degradation. Its superior performance compared to commercial standards suggests immense potential for cost-effective applications in the bio-manufacturing of high-purity fatty acids and biodiesel, offering a greener alternative to traditional chemical processes. Full article

The CRISPR/Cas9 system is a powerful genome-editing tool that is applied in baculovirus engineering. In this study, we present the first report of the AcMNPV genome deletions for bioproduction purposes, using a dual single-guide RNA (sgRNA) CRISPR/Cas9 approach. We used this method to remove nonessential genes for the budded virus and boost recombinant protein yields when applied as BEVS. We show that the co-delivery of two distinct ribonucleoprotein (RNP) complexes, each assembled with a sgRNA and Cas9, into Sf9 insect cells efficiently generated deletions of fragments containing tandem genes in the genome. To evaluate the potential of this method, we assessed the expression of two model proteins, eGFP and HRPc, in insect cells and larvae. The gene deletions had diverse effects on protein expression: some significantly enhanced it while others reduced production. These results indicate that, although the targeted genes are nonessential, their removal can differentially affect recombinant protein yields depending on the host. Notably, HRPC expression increased up to 3.1-fold in Spodoptera frugiperda larvae. These findings validate an effective strategy for developing minimized baculovirus genomes and demonstrate that dual-guide CRISPR/Cas9 editing is a rapid and precise tool for baculovirus genome engineering. Full article

Macrobrachium rosenbergii, commonly known as giant freshwater prawns (GFPs), is an economically and nutritionally important decapod crustacean species in China. Understanding the genetic diversity of selective breeding populations is crucial in breeding plans for selecting genetically diverse broodstocks and maintaining genetic diversity. The genetic structure of six breeding populations (Hefu (HF), Nantaihu No.2 (NTH), Jiaxin (JX), Shufeng (SF), Taiwan (TW), and Guangxi (GX)) of GFP in China was examined using 16 newly developed microsatellite loci and the mitochondrial control region (D-loop). The microsatellite data revealed that all 16 loci have high diversity, with all values of polymorphism information content (PIC) more than 0.5. The average expected heterozygosity (He, 0.89) and the number of alleles (Na, 18.25) of SF were the highest, followed by He (0.89) and Na (14.75) of the JX, and GX has the lowest He (0.83) and Na (11.31). The average PIC value for the six stocks ranged from 0.80 to 0.87. Pairwise comparisons revealed that Fst ranged from 0.03541 to 0.09637 and was significant (p < 0.05) between most populations, indicating from low to moderate genetic differentiation among the six populations. The D-loop analysis identified 114 variable sites and 29 haplotypes, with an average haplotype diversity (Hd) and nucleotide diversity (π) of 0.640 and 0.01247, respectively. Genetic differentiation among the six populations based on the D-loop was from moderate to high, with Fst values of 0.05603–0.80788, and all p < 0.05. This study demonstrates that selective breeding stocks of M. rosenbergii in China show moderate to high genetic diversity and have the potential for further selective breeding, providing a theoretical basis for conserving and utilizing M. rosenbergii genetic resources. Full article

Our previous study demonstrated that an Escherichia coli heterologous secretion expression system, mediated by superfolder green fluorescent protein (sfGFP) mutants, significantly enhances recombinant lipase yield and reduces large-scale production costs. In this study, we identified mScarlet3, a fast-folding fluorescent protein, as another effective mediator of secretion expression in E. coli. A novel lipolytic enzyme, named LipHu6, was identified through sequence alignment. Secretion expression of LipHu6 was achieved by fusing mScarlet3 to either its N- or C-terminus. The specific activity of mScarlet3-LipHu6 reached 669,151.75 U/mmol, slightly surpassing that of LipHu6 alone (646,682.69 U/mmol) and markedly exceeding that of sfGFP_(-15)-LipHu6 (492,432.39 U/mmol). Notably, N-terminal mScarlet3 fusion had no impact on LipHu6 hydrolytic activity toward short-chain p-nitrophenyl fatty acyl esters (C2–C8). In contrast, mScarlet3-LipHu6 exhibited approximately 1.5- and 1.7-fold increases in hydrolytic activity toward p-nitrophenyl palmitate (p-NPP, C16) and p-nitrophenyl stearate (p-NPS, C18), respectively. In conclusion, this study establishes a novel E. coli heterologous secretion expression system mediated by mScarlet3, offering a highly efficient and cost-effective strategy for the large-scale production of lipolytic enzymes. Full article

3,4-Dihydroxy-L-phenylalanine (DOPA) is a promising noncanonical amino acid (ncAA) that introduces novel catechol chemical features into proteins, expanding their functional potential. However, the most common approach to incorporating ncAAs into proteins relies on stop codon suppression, which is often limited by the competition of endogenous translational termination machinery. Here, we employed a special in vitro protein expression system that facilitates the efficiency of DOPA incorporation into proteins by removing essential Class I peptide release factors through targeted degradation. In the absence of both RF1 and RF2, we successfully demonstrated DOPA incorporation at all three stop codons (TAG, TAA, and TGA). By optimizing the concentration of engineered DOPA-specific aminoacyl-tRNA synthetase (DOPARS), DOPA, and DNA template, we achieved a synthesis yield of 2.24 µg of sfGFP with 100% DOPA incorporation in a 20 μL reaction system. DOPARS exhibited a dissociation constant (Kd) of 11.7 μM for DOPA but showed no detectable binding to its native counterpart, tyrosine. Additionally, DOPA was successfully incorporated into a reverse transcriptase, which interfered with its activity. This system demonstrates a fast and efficient approach for precise DOPA incorporation into proteins, paving the way for advanced protein engineering applications. Full article

Tetrahydrofolate (THF), the biologically active form of folate, serves as a crucial carrier of one-carbon units essential for synthesizing cellular components such as amino acids and purine nucleotides in vivo. It also acts as an important precursor for the production of pharmaceuticals, including folinate and L-5-methyltetrahydrofolate (L-5-MTHF). In this study, we developed an efficient enzyme cascade system for the production tetrahydrofolate from folate, incorporating NADPH recycling, and explored its application in the synthesis of L-5-MTHF, a derivative of tetrahydrofolate. To achieve this, we first screened dihydrofolate reductases (DHFRs) from various organisms, identifying SmDHFR from Serratia marcescens as the enzyme with the highest catalytic activity. We then conducted a comparative analysis of formate dehydrogenases (FDHs) from different sources, successfully establishing an NADPH recycling system. To further enhance biocatalytic efficiency, we optimized key reaction parameters, including temperature, pH, enzyme ratio, and substrate concentration. To address the challenge of pH mismatch in dual-enzyme reactions, we employed an enzymatic microenvironment regulation strategy. This involved covalently conjugating SmDHFR with a superfolder green fluorescent protein mutant carrying 30 surface negative charges (−30sfGFP), using the SpyCatcher/SpyTag system. This modification resulted in a 2.16-fold increase in tetrahydrofolate production, achieving a final yield of 4223.4 µM. Finally, we extended the application of this tetrahydrofolate synthesis system to establish an enzyme cascade for L-5-MTHF production with NADH recycling. By incorporating methylenetetrahydrofolate reductase (MTHFR), we successfully produced 389.8 μM of L-5-MTHF from folate and formaldehyde. This work provides a novel and efficient pathway for the biosynthesis of L-5-MTHF and highlights the potential of enzyme cascade systems in the production of tetrahydrofolate-derived compounds. Full article

Colored proteins play an important role in synthetic biology research, providing a systematic labeling tool for visualizing microscopic biological activities in vivo. They can exhibit visible colors to the naked eye under natural light, and some of them are well-known fluorescent proteins. Here, several colored proteins were taken into consideration for acting as biocolorants in Escherichia coli, including green fluorescent proteins (eGFP and sfGFP), a red fluorescent protein (mKate2), and three chromoproteins (GfasPurple, AmilCP, and AeBlue). All of them can significantly change the colors of their bacterial colonies. The color of GfasPurple was much more stable after the heat treatments at 65 °C with 75% or 95% ethanol. In addition, several factors commonly occurring under natural conditions that lead to color dissolution, such as heat, ethanol, H₂O₂, vitamin C, acid, and alkali treatments, were further tested on GfasPurple. Visual observation and absorption spectroscopy analysis results showed an excellent tolerance of GfasPurple against these unfriendly conditions. GfasPurple could withstand temperatures of 65 °C for 2 h or 70 °C for 1 h in aqueous solutions, but it fades rapidly in 50% ethanol. The color of GfasPurple is more stable in 80% ethanol than in 50% ethanol, which could be attributed to its poor solubility in high-concentration ethanol. The visible light absorption curves of GfasPurple were basically not affected by physiological concentrations of vitamin C or H₂O₂, but reversible effects of high-concentration H₂O₂ were found. GfasPurple maintains its color within the pH range of 7–11; the chromophore of GfasPurple will suffer irreversible damage when pH is up to thirteen or as low as three. These results suggest that GfasPurple is an excellent biocolorant far beyond its application in prokaryotes. Furthermore, GfasPurple variants created via mutagenesis expanded the color library of chromoproteins, which have a potential value in the color manipulation of living organisms. Full article

Nanobodies have gained attention as potential therapeutic and diagnostic agents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to their ability to bind and neutralize the virus. However, rapid, scalable, and robust production of nanobodies for SARS-CoV-2 remains a crucial challenge. In this study, we developed a visual and high-efficiency biomanufacturing method for nanobodies with Escherichia coli by fusing the super-folder green fluorescent protein (sfGFP) to the N-terminus or C-terminus of the nanobody. Several receptor-binding domain (RBD)-specific nanobodies of the SARS-CoV-2 spike protein (S) were secreted onto the surface of E. coli cells and even into the culture medium, including Fu2, ANTE, mNb6, MR3-MR3, and n3113.1. The nanobodies secreted by E. coli retained equal activity as prior research, regardless of whether sfGFP was removed. Since some of the nanobodies bound to different regions of the RBD, we combined two nanobodies to improve the affinity. Fu2-sfGFP-ANTE was constructed to be bispecific for the RBD, and the bispecific nanobody exhibited significantly higher affinity than Fu2 (35.0-fold), ANTE (7.3-fold), and the combination of the two nanobodies (3.3-fold). Notably, Fu2-sfGFP-ANTE can be normally secreted into the culture medium and outer membrane. The novel nanobody production system enhances the efficiency of nanobody expression and streamlines the downstream purification process, enabling large-scale, cost-effective nanobody production. In addition, E. coli cells secreting the nanobodies on their surface facilitates screening and characterization of antigen-binding clones. Full article

Vibrio parahaemolyticus (V. parahaemolyticus) is a significant concern, as it can cause severe infections and hemolytic trauma. Given its prevalence in seawater and coastal seafood, it poses a substantial risk as a foodborne pathogen. Biosensor-based detection technology has been continuously evolving, and toehold switches have emerged as a promising area within it, especially in the detection of RNA viruses. Here, we have developed a cell-free toehold switch sensor for V. parahaemolyticus detection. Traditional toehold switch detection methods usually use green fluorescent protein (GFP) or enzyme LacZ as the output signal, with an incubation time as long as 2 h, and are also mainly applied to the detection of RNA viruses. In this study, we introduced a novel, artificially designed luciferase (LuxSit-i) as an output signal and constructed toehold switches with two different output signals (sfGFP, LuxSit-i), aimed at reducing the incubation time of toehold switches. Moreover, to further improve the detection process, we separately utilize recombinase polymerase amplification (RPA) and nucleic acid sequence-based amplification (NASBA) to amplify dead and live bacterial suspensions for detection and attempt to distinguish between dead and live bacteria. This study provided a convenient, rapid, and accurate method for the on-site detection of V. parahaemolyticus, especially beneficial for resource-limited settings. By eliminating the requirement for specialized facilities and personnel, this system has the potential to be a valuable tool in improving public health responses, especially in developing regions. Full article

Small regulatory RNAs (sRNA) have been shown to play a large role in the management of stress responses in Escherichia coli and other bacteria. Upon fluctuations in nutrient availability and exposure to antimicrobials and superoxide-generating agents, the MicF sRNA in E. coli has been shown to regulate a small set of genes involved in the management of membrane permeability. Currently, it is unknown whether MicF acts on other processes to mediate the response to these agents. Using an sRNA interaction prediction tool, we identified genes in E. coli that are potentially regulated by MicF. Through subsequent analysis using a sfGFP-based reporter–gene fusion, we have validated two novel targets of MicF regulation: ObgE, a GTPase crucial for chromosome partitioning, and SeqA, a negative modulator of DNA replication. Importantly, the interaction between MicF and these target mRNAs is contingent upon the presence of the RNA chaperone protein, Hfq. Furthermore, our findings affirm the role of MicF’s conserved 5’ seed pairing region in initiating these regulatory interactions. Our study suggests that, beyond its established role in membrane permeability management, MicF exerts control over chromosome dynamics in response to distinct environmental cues, implicating a more multifaceted regulatory function in bacterial stress adaptation. Full article

In recent years, various technologies have emerged for the imaging of chromatin loci in living cells via catalytically inactive Cas9 (dCas9). These technologies facilitate a deeper understanding of the mechanisms behind the chromatin dynamics and provide valuable kinetic data that could not have previously been obtained via FISH applied to fixed cells. However, such technologies are relatively complicated, as they involve the expression of several chimeric proteins as well as sgRNAs targeting the visualized loci, a process that entails many technical subtleties. Therefore, the effectiveness in visualizing a specific target locus may be quite low. In this study, we directly compared two versions of a previously published CRISPR-Sirius method based on the use of sgRNAs containing eight MS2 or PP7 stem loops and the expression of MCP or PCP fused to fluorescent proteins. We assessed the visualization efficiency for several unique genomic loci by comparing the two approaches in delivering sgRNA genes (transient transfection and lentiviral transduction), as well as two CRISPR-Sirius versions (with PCP and with MCP). The efficiency of visualization varied among the loci, and not all loci could be visualized. However, the MCP-sfGFP version provided more efficient visualization in terms of the number of cells with signals than PCP-sfGFP for all tested loci. We also showed that lentiviral transduction was more efficient in locus imaging than transient transfection for both CRISPR-Sirius systems. Most of the target loci in our study were located at the borders of topologically associating domains, and we defined a set of TAD borders that could be effectively visualized using the MCP-sfGFP version of the CRISPR-Sirius system. Altogether, our study validates the use of the CRISPR-Sirius technology for live-cell visualization and highlights various technical details that should be considered when using this method. Full article

Promyelocytic leukemia (PML) nuclear bodies (PML-NBs) are core–shell-type membrane-less organelles typically found in the nucleus of mammalian somatic cells but are absent in mouse oocytes. Here, we deliberately induced the assembly of PML-NBs by injecting mRNA encoding human PML protein (hPML VI -sfGFP) into oocytes and investigated their impact on fertilization in which oocyte/embryos undergo multiple types of stresses. Following nuclear membrane breakdown, preassembled hPML VI -sfGFP mRNA-derived PML-NBs (hmdPML-NBs) persisted in the cytoplasm of oocytes, forming less-soluble debris, particularly under stress. Parthenogenetic embryos that successfully formed pronuclei were capable of removing preassembled hmdPML-NBs from the cytoplasm while forming new hmdPML-NBs in the pronucleus. These observations highlight the beneficial aspect of the PML-NB-free nucleoplasmic environment and suggest that the ability to eliminate unnecessary materials in the cytoplasm of metaphase oocytes serves as a potential indicator of the oocyte quality. Full article

Alpha-ketoglutaric acid (α-KG), as an intermediate product of the tricarboxylic acid cycle, plays a crucial role in peptide and amino acid synthesis. In order to reduce costs and improve efficiency in the oxidative production of α-ketoglutaric acid, this study successfully synthesized and expressed L-glutamate oxidase (LGOX^Str) from Streptomyces viridosporus R111 and catalase (KatG^Esc) from Escherichia coli H736. Two immobilization methods and the conditions for one-step whole-cell catalysis of α-ketoglutaric acid were investigated. α-Ketoglutaric acid has broad applications in the pharmaceutical, food, and chemical industries. The specific research results are as follows: (1) By fusing the sfGFP tag, L-glutamate oxidase (LGOX^Str r) and catalase (KatG^Esc) were successfully anchored to the outer membrane of Escherichia coli cells, achieving one-step whole-cell catalysis of α-ketoglutaric acid with a conversion efficiency of up to 75%. (2) Through the co-immobilization of LGOX^Str and KatG^Esc, optimization of the preparation parameters of immobilized cells, and exploration of the immobilization method using E.coli@ZIF-8, immobilized cells with conversion rates of over 60% were obtained even after 10 cycles of reuse. Under the optimal conditions, the production rate of α-ketoglutaric acid reached 96.7% in a 12 h reaction, which is 1.1 times that of E. coli@SA and 1.29 times that of free cells. Full article