Search Results (82)

In view of the technical bottleneck of microbial dynamic monitoring during the solid-state fermentation of traditional Baijiu, this study introduced green fluorescent protein (GFP) labeling technology into the dominant Saccharomyces cerevisiae of Jiang-flavored Baijiu to construct the chromosomal integration engineering strain named FSC01. By designing an integrated recombinant plasmid containing the GFP gene and the geneticmycin resistance gene, an engineered strain that stably expresses fluorescent proteins was obtained by electroconversion. Flow cytometry verification showed that FSC01 showed excellent linear responses in the pure microbial system (R² = 0.998) and the complex matrix of Baijiu jiupei (R² = 0.981), with a detection limit of 10² cells/mL, and the detection cycle was shortened to 10 min. Solid-state fermentation simulation experiments show that the inoculation volume of FSC01 of 10⁵ cells/kg can not only ensure the effective identification of fluorescence signals, but also does not significantly interfere with the growth and growth patterns of the original yeast (p > 0.05), which is highly consistent with the results of the traditional plate counting method. Dynamic monitoring shows that Saccharomyces cerevisiae during fermentation presents a typical succession pattern of “increase first and then decrease”, reaching a peak on the 7th day (1.2 × 10⁷ cells/g), which is positively correlated with the base alcohol yield rate (26.7%). Compared with metagenomic (72 h) and PMA-qPCR (4 h) methods, this technology breaks through the limitations of specificity and timeliness of live bacteria detection, and provides a single-cell-level dynamic analysis tool for the digitization of traditional brewing processes. In the future, it will be expanded to monitor key functional microorganisms such as lactic acid bacteria through a multi-color fluorescent labeling system, and optimized pretreatment to eliminate starch granule interference, and promote the in-depth application of synthetic biology technology in the traditional fermentation industry. Full article

Osteoporosis is a systemic skeletal disease that severely impairs the health of the elderly population. The interaction between the receptor activator of the NF-κB ligand (RANKL) and its receptor RANK is critical for osteoclast differentiation and function. Therefore, targeting the RANKL/RANK interaction represents a promising strategy for osteoporosis. In this study, we employed a newly established yeast two-hybrid system based on RANKL/RANK interaction and identified IMB-R38, a novel benzamide compound that dose-dependently blocked RANKL/RANK interaction by inhibiting the growth of AH109 cells harboring pAD-RANKL/pBD-RANK plasmids in quadruple-dropout medium. IMB-R38 significantly suppressed osteoclast differentiation, disrupted F-actin ring formation, and downregulated the expression of osteoclast-specific genes, including NFATc1 and MMP9 in RANKL-induced RAW264.7 macrophages. IMB-R38 also promoted osteoblast differentiation by upregulating the expression of osteogenic genes. Importantly, in a dexamethasone (DXM)-induced osteoporotic zebrafish model, IMB-R38 significantly increased bone mineralization, with anti-osteoporosis efficacy superior to that of alendronate sodium (Alen). RT-qPCR assays showed that IMB-R38 significantly upregulated the mRNA expression of osteogenesis genes (Bmp2, Runx2a, Runx2b, Sp7, Alp, and Oc) while markedly downregulating that of the osteoclastogenesis genes (Mmp9, Mmp13, and Mmp2) compared with the DXM group. Mechanistically, an SPR assay confirmed that IMB-R38 directly binds with RANK but not RANKL to disrupt RANKL/RANK interaction. Furthermore, Asp168 of RANK was identified as a key amino acid that mediates both RANKL interaction and IMB-R38 binding. The inhibition of RANKL/RANK by IMB-R38 suppressed JNK phosphorylation and, consequently, osteoclast differentiation and function. Collectively, our findings identify IMB-R38 as a novel RANKL/RANK inhibitor with therapeutic potential for osteoporosis through its regulation of bone metabolism. Full article

Microsomal fractions from yeast Δale1 cells harbouring the empty plasmid pYES2/CT and from yeast cells overexpressing PtATS2a (Phatr3_J11916) or PtATS2b (Phatr3_J43099) were used in the studies. When sn-1-18:1-LPA and [¹⁴C]16:0-CoA were used as exogenous substrates, both PtATS2a and PtATS2b showed the highest activity at 23 °C in the range of temperatures tested from 10 to 60 °C. Both enzymes showed the highest activity in alkaline pH. For PtATS2a, it was pH 10 while for PtATS2b, it was pH 11. At pH 6 and pH 12, the activities of both enzymes were very low. The calcium ions at concentrations of 0.05–1 mM drastically decreased the activity of both enzymes. The magnesium ions at a concentration of 0.05 mM had a little effect on the activity of both enzymes, while higher concentrations (0.5 mM and 1 mM) significantly inhibited their activity. To study the substrate specificity, seventeen different acyl-CoAs in combinations with sn-1-[¹⁴C]18:1-LPA were used. PtATS2a showed the highest preference for 18:4-CoA n-3 while PtATS2b for 18:1-CoA. The pattern of utilisation of other acyl-CoAs tested also differed between the two enzymes. The presented studies, for the first time, characterised LPAAT type enzymes from diatoms, organisms that naturally produced very-long-chain polyunsaturated fatty acids (VLC-PUFA). Full article

In the present research, metal–organic framework-5 (MOF-5) was synthesized and loaded with zerumbone (ZER@MOF-5), followed by the evaluation of its anticancer, antibacterial, antifungal, DNA-binding, and free radical scavenging potentials. The synthesized nanoparticles were characterized using X-ray diffraction, ultraviolet–visible spectroscopy, Fourier-transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, and scanning electron microscopy. The in vitro anticancer activity of ZER@MOF-5 was studied in a human breast cancer cell line (MCF-7) using the CCK-8 assay. The interaction of ZER@MOF-5 with pBR322 plasmid DNA was assessed by gel electrophoresis. The antimicrobial effect of ZER@MOF-5 was examined in gram-positive and gram-negative bacterial strains and yeast strains using the microdilution method. The free radical scavenging activity was assessed using the DPPH assay. Cytotoxicity assay revealed a notable enhancement in the anticancer activity of zerumbone upon its encapsulation into MOF-5. The IC₅₀ value for ZER@MOF-5 was found to be 57.33 µg/mL, which was lower than that of free zerumbone (IC₅₀: 89.58 µg/mL). The results of the DNA-binding experiment indicate that ZER@MOF-5 can bind to target DNA and cause a conformational change in DNA. The results of the antibacterial activity experiment showed that the antibacterial ability of ZER@MOF-5 was limited compared to free zerumbone. The results of the DPPH assay demonstrated that the antioxidant activity of free zerumbone was higher than that of ZER@MOF-5. MOFs encapsulate compounds within their porous crystalline structure, which leads to prolonged circulation time compared to single ligands. Although the unique structure of MOFs may limit their antibacterial and antioxidant activity in the short term, it may increase therapeutic efficacy in the long term. However, to fully understand the long-term antibacterial and antioxidant effects of the ZER@MOF-5, further comprehensive in vitro and in vivo experiments are necessary. This finding indicates that the MOF-5 could potentially be an impressive carrier for the oral administration of zerumbone. Full article

We have generated a vector that enables the removal of plasmids coding for truncated proteins. This vector expresses a protein of interest in the yeast Saccharomyces cerevisiae from a galactose-inducible promoter. The gene of interest is fused in-frame to a downstream sequence coding for phosphoribosylanthranilate isomerase (PRAI), which catalyses the third step in tryptophan biosynthesis. As a consequence, only the full-length protein of interest renders the host cell tryptophan prototrophic, allowing for selection against cells expressing truncated proteins. Our proof-of-principle study demonstrates that PRAI is functional when fused C-terminally to a protein, robustly rendering cells tryptophan prototrophic. The N-terminal GST tag and C-terminal myc tag allow for tag-mediated protein purification, co-precipitation studies, determination of relative expression levels, as well as validation of full-length expression of the protein via Western blotting. Full article

Cellulosic biomass is considered an important and sustainable source of renewable energy, which needs a complex mixture of different enzymes for its degradation. After amylase, cellulases are the second most important enzymes, gain more importance due to their broad range of applications at the industrial level, and are considered more economical and environmentally friendly; researchers have focused more on the production of cellulase with its higher expression rate and low cost. Pichia pastoris, a methylotrophic yeast strain, has a more effective and well-established system for the production of heterologous proteins, particularly for industrial enzymes. Moreover, its readily achievable high-density fermentation, high capacity for protein secretion, tractable genetic modifications, typical post-transcriptional modifications, and strong regulated promoters makes it superior to other expression systems. In this review, we address the P. pastoris expression system including protein expression platforms, plasmids, and cellular metabolism quantification as a potential candidate for heterologous protein production, particularly for cellulase enzymes. Full article

Fluorine accounts for 0.3 g/kg of the Earth’s crust, being widely distributed in the environment as fluoride. The toxic effects of this anion in humans and other organisms have been known for a long time. Fluoride has been reported to alter several cellular processes although the mechanisms involved are largely unknown. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes that hydrolyze inorganic pyrophosphate (PPi), a metabolite generated from ATP. In Saccharomyces cerevisiae, the enzyme responsible for PPi hydrolysis in the cytosol (IPP1) is strongly inhibited by fluoride in vitro. The essentiality of IPP1 for growth has been previously demonstrated using YPC3, a yeast mutant with conditional expression of the corresponding gene. Here, YPC3 was used to generate cells that tolerate high concentrations of fluoride by (a) the overexpression of IPP1 or its human ortholog, or (b) the substitution of IPP1 by the fluoride-insensitive PPase from Streptococcus mutans. The results obtained suggest that maintaining appropriate levels of PPase activity in the cytosol is essential for the adaptation of S. cerevisiae to high fluoride concentrations. The increase in fluoride tolerance allows YPC3 cells transformed with suitable plasmids to be selected on rich non-selective medium supplemented with this anion. Full article

Reverse genetics is a useful tool for studying viruses and developing vaccines for coronaviruses. However, constructing and manipulating the coronavirus genome in Escherichia coli can be time-consuming and challenging due to its large size and instability. Homologous recombination, a genetic manipulation mechanism found in organisms, is essential for DNA repair, gene recombination, and genetic engineering. In yeast, particularly Saccharomyces cerevisiae, homologous recombination technology is commonly used for constructing gene expression plasmids and genome editing. In this study, we successfully split and spliced a 30 kb viral genome fragment using yeast homologous recombination. By optimizing the program parameters, such as homologous arm lengths and fragment-to-vector ratios, we achieved a splicing efficiency of up to 97.9%. The optimal parameters selected were a 60 bp homologous sequence size and a vector fragment ratio of 1:2:2:2:2:2 for yeast homologous recombination of large DNA fragments. Full article

Background/Objectives: DNA vaccines are rapidly produced and adaptable to different pathogens, but they face considerable challenges regarding stability and delivery to the cellular target. Thus, effective delivery methods are essential for the success of these vaccines. Here, we evaluated the efficacy of capsules derived from the cell wall of the yeast Pichia pastoris as a delivery system for DNA vaccines. Methods: The capsules were extracted from the yeast Pichia pastoris strain GS115, previously grown in a YPD medium. pVAX1 expression vector was adopted to evaluate the DNA vaccine insertion and delivery. Three encapsulation protocols were tested to identify the most effective in internalizing the plasmid. The presence of plasmids inside the capsules was confirmed by fluorescence microscopy, and the encapsulation efficiency was calculated by the difference between the initial concentration of DNA used for insertion and the concentration of unencapsulated DNA contained in the supernatant. The capsules were subjected to different temperatures to evaluate their thermostability and were co-cultured with macrophages for phagocytosis analysis. HEK-293T cells were adopted to assess the cytotoxicity levels by MTT assay. Results: The microscopy results indicated that the macrophages successfully phagocytosed the capsules. Among the protocols tested for encapsulation, the one with 2% polyethylenimine for internalization showed the highest efficiency, with an encapsulation rate above 80%. However, the vaccine capsules obtained with the protocol that used 5% NaCl showed better thermal stability and encapsulation efficiency above 63% without induction of cell viability loss in HEK 293T. Conclusions: We successfully described a vaccine delivery system using yeast capsules derived from Pichia pastoris, demonstrating its potential for DNA vaccine delivery for the first time. Additional studies will be needed to characterize and improve this delivery strategy. Full article

Background: Introducing unnatural base pairs into a natural, double-stranded DNA construct is a powerful tool within synthetic biology. Accordingly, the ability to detect these unnatural base pairs has many applications, including the study and detection of semisynthetic organisms. Objective and Methods: The work described here aimed to select human antibodies for the specific recognition of Hirao’s base pair dDs–dPn in various natural DNA contexts by using a combination of phage and yeast display technologies. Results: We selected a total of six antibodies in yeast-displayed scFv format, and further tested three of these antibodies in soluble form as minibodies and IgGs. We also describe an assay that can be used to detect plasmids containing dDs–dPn pair. Conclusions: Our antibodies did not afford the desired specificity or sensitivity for detection of a single unnatural base pair among thousands of natural. However, our data indicate not only that such detection is possible but also that these antibodies may be candidates for further affinity and specificity maturation. Full article

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that infect pigs’ intestinal epithelial cells, causing high morbidity and mortality. Due to the rapid mutation of PEDV, vaccine efficacy is uncertain, prompting exploration of alternative treatments. Nanobodies, also known as variable heavy chain domains of heavy chain-only antibodies (VHHs), offer significant potential in biomedical applications due to their small size and high specificity. In this study, yeast two-hybrid technology was employed to screen for eight specific VHH sequences targeting the PEDV S protein from a synthetically constructed nanobody yeast library. The VHH genes were then cloned into expression plasmids for recombinant protein production, and the resulting VHHs (termed PEDV S-VHHs) were purified. Indirect immunofluorescence assay (IFA) and Western blotting analysis confirmed that these VHHs specifically bind to both PEDV and its S protein. Neutralization assays demonstrated that seven PEDV S-VHHs exhibited potent neutralizing activity against PEDV. Additionally, a combination of these seven antibodies showed enhanced antiviral effects. Preliminary predictions were also made regarding the binding sites between these VHHs and PEDV. The PEDV S-VHHs described in this study hold potential as candidates for the prevention and treatment of PEDV infection. Full article

Retrotransposons are mobile DNA elements that are more active with increasing age and exacerbate aging phenotypes in multiple species. We previously reported an unexpected extension of chronological lifespan in the yeast, Saccharomyces paradoxus, due to the presence of Ty1 retrotransposons when cells were aged under conditions of mild stress. In this study, we tested a subset of genes identified by RNA-seq to be differentially expressed in S. paradoxus strains with a high-copy number of Ty1 retrotransposons compared with a strain with no retrotransposons and additional candidate genes for their contribution to lifespan extension when cells were exposed to a moderate dose of hydroxyurea (HU). Deletion of ADE8, NCS2, or TRM9 prevented lifespan extension, while deletion of CDD1, HAC1, or IRE1 partially prevented lifespan extension. Genes overexpressed in high-copy Ty1 strains did not typically have Ty1 insertions in their promoter regions. We found that silencing genomic copies of Ty1 prevented lifespan extension, while expression of Ty1 from a high-copy plasmid extended lifespan in medium with HU or synthetic medium. These results indicate that cells adapt to expression of retrotransposons by changing gene expression in a manner that can better prepare them to remain healthy under mild stress. Full article

Recombination hotspot-activating DNA sites (e.g., M26, CCAAT, Oligo-C) and their binding proteins (e.g., Atf1-Pcr1 heterodimer; Php2-Php3-Php5 complex, Rst2, Prdm9) regulate the distribution of Spo11 (Rec12)-initiated meiotic recombination. We sought to create 14 different candidate regulatory DNA sites via bp substitutions in the ade6 gene of Schizosaccharomyces pombe. We used a fission yeast-optimized CRISPR-Cas9 system (SpEDIT) and 196 bp-long dsDNA templates with centrally located bp substitutions designed to ablate the genomic PAM site, create specific 15 bp-long DNA sequences, and introduce a stop codon. After co-transformation with a plasmid that encoded both the guide RNA and Cas9 enzyme, about one-third of colonies had a phenotype diagnostic for DNA sequence changes at ade6. PCR diagnostics and DNA sequencing revealed a diverse collection of alterations at the target locus, including: (A) complete or (B) partial template-directed substitutions; (C) non-homologous end joinings; (D) duplications; (E) bp mutations, and (F) insertions of ectopic DNA. We concluded that SpEDIT can be used successfully to generate a diverse collection of DNA sequence elements within a reporter gene of interest. However, its utility is complicated by low efficiency, incomplete template-directed repair events, and undesired alterations to the target locus. Full article

The PHO91 and PHO87 are parts of yeast’s phosphate metabolism system. We investigated the influence of PHO91 deletion on the methanol utilization as a sole carbon source in Ogataea parapolymorpha through comparison of wet biomass, protein content, methanol oxidase (MOX) activity and acid-soluble and acid-insoluble inorganic polyphosphate (polyP) content, while growing on methanol and without any carbon source other than trace amounts from the yeast extract. The Δpho91 strain has a lack of wet biomass, protein content and MOX activity while grown on methanol (as a ΔMOX strain) and has the same numbers as a WT strain in media without methanol. We found two ways of recovering methanol utilizing capabilities for Δpho91 mutant—either adding MOX on plasmid under constitutive promoter control, or adding a Δpho87 mutation. Both Δpho91 + MOX and Δpho91Δpho87 strains showed all the parameters as a WT strain on methanol as a sole carbon source and without methanol addition. Further, we compare the polyP content of the strains in methanol media, media without a sole carbon source and media with glucose. All of the WT, Δpho91 + MOX and Δpho91Δpho87 strains, capable of utilizing methanol, have the P_i, acid-soluble and acid-insoluble polyP at the same levels with some variations. The ΔMOX and Δpho91 strains, despite both not being able to utilize methanol, showed very different levels of P_i and polyP’s, which can be interpreted as different mechanisms of the inner cell, leading to a loss of capability utilizing methanol in both of these strains. Full article

In the context of the growing concern regarding the appearance and spread of emerging pathogens with high resistance to chemically synthetized biocides, the development of new agents for crops and human protection has become an emergency. In this context, the yeasts present a huge potential as eco-friendly agents due to their widespread nature in various habitats and to their wide range of antagonistic mechanisms. The present review focuses on some of the major yeast antimicrobial mechanisms, their molecular basis and practical applications in biocontrol and biomedicine. The synthesis of killer toxins, encoded by dsRNA virus-like particles, dsDNA plasmids or chromosomal genes, is encountered in a wide range of yeast species from nature and industry and can affect the development of phytopathogenic fungi and other yeast strains, as well as human pathogenic bacteria. The group of the “red yeasts” is gaining more interest over the last years, not only as natural producers of carotenoids and rhodotorulic acid with active role in cell protection against the oxidative stress, but also due to their ability to inhibit the growth of pathogenic yeasts, fungi and bacteria using these compounds and the mechanism of competition for nutritive substrate. Finally, the biosurfactants produced by yeasts characterized by high stability, specificity and biodegrability have proven abilities to inhibit phytopathogenic fungi growth and mycelia formation and to act as efficient antibacterial and antibiofilm formation agents for biomedicine. In conclusion, the antimicrobial activity of yeasts represents a direction of research with numerous possibilities of bioeconomic valorization as innovative strategies to combat pathogenic microorganisms. Full article