Search Results (15)

In this study, the probiotic yeast Saccharomyces boulardii was engineered to secrete the antiviral lectin griffithsin. Twelve genetic tools with the griffithsin gene were cloned into the vector pSF-TEF1-URA3 and introduced into S. boulardii. In the recombinant strains, a 16.9 kDa band was detected using SDS-PAGE and further recognized by griffithsin antibody with Western blotting. S. boulardii strains FM, FT, HC, and HE with a high yield of griffithsin were acquired for property characterization in vitro. The four recombinant strains displayed a similar growth pattern to that of the control strains, while their morphological characteristics had changed according to scanning electron microscopy. In simulated gastrointestinal digestive fluids, the survival rates of S. boulardii FM, FT, and HC were significantly decreased (86.32 ± 1.49% to 95.36 ± 1.94%) compared with those of the control strains, with survival rates between 95.88 ± 0.00% and 98.74 ± 1.97%. The hydrophobicity of S. boulardii FM, the strain with the highest griffithsin production, was significantly increased to 21.89 ± 1.07%, and it exhibited a reduced auto-aggregation rate (57.64 ± 2.61%). Finally, Vero cells infected with porcine epidemic diarrhea virus (PEDV) were used to evaluate the strains’ antiviral activity, and the rate at which S. boulardii FM inhibited PEDV reached 131.36 ± 1.06%, which was significantly higher than that of the control group. Full article

The CRISPR/Cas9 system has become a popular approach to genome editing. Compared with the plasmid-dependent CRISPR system, the ribonucleoprotein (RNP) complex formed by the in vitro assembly of Cas9 and single-guide RNA (sgRNA) has many advantages. However, only a few examples have been reported and the editing efficiency has been relatively low. In this study, we developed and optimized an RNP-mediated CRISPR/Cas9 genome editing system for the monokaryotic strain L1 from the Ganoderma lucidum cultivar ‘Hunong No. 1’. On selective media containing 5-fluoroorotic acid (5-FOA), the targeting efficiency of the genomic editing reached 100%. The editing efficiency of the orotidine 5′-monophosphate decarboxylase gene (ura3) was greater than 35 mutants/10⁷ protoplasts, surpassing the previously reported G. lucidum CRISPR systems. Through insertion or substitution, 35 mutants introduced new sequences of 10–569 bp near the cleavage site of ura3 in the L1 genome, and the introduced sequences of 22 mutants (62.9%) were derived from the L1 genome itself. Among the 90 mutants, 85 mutants (94.4%) repaired DNA double-strand breaks (DSBs) through non-homologous end joining (NHEJ), and five mutants (5.6%) through microhomology-mediated end joining (MMEJ). This study revealed the repair characteristics of DSBs induced by RNA-programmed nuclease Cas9. Moreover, the G. lucidum genes cyp512a3 and cyp5359n1 have been edited using this system. This study is of significant importance for the targeted breeding and synthetic metabolic regulation of G. lucidum. Full article

Sugar transport from the source leaf to the sink organ is critical for seed development and crop yield, as well as for responding to abiotic stress. SWEETs (sugar will eventually be exported transporters) mediate sugar efflux into the reproductive sink and are therefore considered key candidate proteins for sugar unloading during seed development. However, the specific mechanism underlying the sugar unloading to seeds in Camellia oleifera remains elusive. Here, we identified a SWEET gene named CoSWEET10, which belongs to Clade III and has high expression levels in the seeds of C. oleifera. CoSWEET10 is a plasma membrane-localized protein. The complementation assay of CoSWEET10 in SUSY7/ura3 and EBY.VW4000 yeast strains showed that CoSWEET10 has the ability to transport sucrose, glucose, and fructose. Through the C. oleifera seeds in vitro culture, we found that the expression of CoSWEET10 can be induced by hexose and sucrose, and especially glucose. By generating the restoration lines of CoSWEET10 in Arabidopsis atsweet10, we found that CoSWEET10 restored the seed defect phenotype of the mutant by regulating soluble sugar accumulation and increased plant drought tolerance. Collectively, our study demonstrates that CoSWEET10 plays a dual role in promoting seed development and enhancing plant drought resistance as a sucrose and hexose transporter. Full article

Contamination of water by microcystins is a global problem. These potent hepatotoxins demand constant monitoring and control methods in potable water. Promising approaches to reduce contamination risks have focused on natural microcystin biodegradation led by enzymes encoded by the mlrABCD genes. The first enzyme of this system (mlrA) linearizes microcystin structure, reducing toxicity and stability. Heterologous expression of mlrA in different microorganisms may enhance its production and activity, promote additional knowledge on the enzyme, and support feasible applications. In this context, we intended to express the mlrA gene from Sphingosinicella microcystinivorans B9 in an industrial Saccharomyces cerevisiae strain as an innovative biological alternative to degrade microcystins. The mlrA gene was codon-optimized for expression in yeast, and either expressed from a plasmid or through chromosomal integration at the URA3 locus. Recombinant and wild yeasts were cultivated in medium contaminated with microcystins, and the toxin content was analyzed during growth. Whereas no difference in microcystins content was observed in cultivation with the chromosomally integrated strain, the yeast strain hosting the mlrA expression plasmid reduced 83% of toxins within 120 h of cultivation. Our results show microcystinase A expressed by industrial yeast strains as a viable option for practical applications in water treatment. Full article

An effective selection marker is necessary for genetic engineering and functional genomics research in the post-genomic era. Isaria javanica is an important entomopathogenic fungus with a broad host range and prospective biocontrol potentials. Given that no antibiotic marker is available currently in this fungus, developing an effective selection marker is necessary. In this study, by applying overlap PCR and split-marker deletion strategy, combining PEG-mediated protoplasm transformation method, the uridine auxotrophy gene (ura3) in the I. javanica genome was knocked out. Then, using this transformation system, the pH response transcription factor gene (IjpacC) was disrupted successfully. Loss of IjpacC gene results in an obvious decrease in conidial production, but little impact on mycelial growth. The virulence of the ΔIjpacC mutant on caterpillars is similar to that of the wild-type strain. RT-qPCR detection shows that expression level of an acidic-expressed S53 gene (IF1G_06234) in ΔIjpacC mutant is more significantly upregulated than in the wild-type strain during the fungal infection on caterpillars. Our results indicate that a markerless transformation system based upon complementation of uridine auxotrophy is successfully developed in I. javanica, which is useful for exploring gene function and for genetic engineering to enhance biological control potential of the fungus. Full article

Chitosan is a functional ingredient that is widely used in food chemistry as an emulsifier, flocculant, antioxidant, or preservative. Chitin deacetylases (CDAs) can catalyze the hydrolysis of acetyl groups, making them useful in the clean production of chitosan. However, the high inactivity of crystalline chitin catalyzed by CDAs has been regarded as the technical bottleneck of crystalline chitin deacetylation. Here, we mined the AsCDA gene from the genome of Acinetobacter schindleri MCDA01 and identified a member of the uraD_N-term-dom superfamily, which was a novel chitin deacetylase with the highest deacetylation activity. The AsCDA gene was expressed in Escherichia coli BL21 by IPTG induction, whose activity to colloidal chitin, α-chitin, and β-chitin reached 478.96 U/mg, 397.07 U/mg, and 133.27 U/mg, respectively. In 12 h, the enzymatic hydrolysis of AsCDA removed 63.05% of the acetyl groups from α-chitin to prepare industrial chitosan with a degree of deacetylation higher than 85%. AsCDA, as a potent chitin decomposer in the production of chitosan, plays a positive role in the upgrading of the chitosan industry and the value-added utilization of chitin biological resources. Full article

In eukaryotic cells, loosening of chromatin causes changes in transcription and DNA replication. The artificial conversion of tightly packed chromatin (heterochromatin) to loosely packed chromatin (euchromatin) enables gene expression and regulates cell differentiation. Although some chemicals convert chromatin structures through histone modifications, they lack sequence specificity. This study attempted to establish a novel technology for inducing chromatin loosening in target regions of Saccharomyces cerevisiae. We focused on histone acetylation, which is one of the mechanisms of euchromatin induction. The sequence-recognizing ability of the dead Cas9 (dCas9) and guide RNA (gRNA) complex was used to promote histone acetylation at a targeted genomic locus. We constructed a plasmid to produce a fusion protein consisting of dCas9 and histone acetyltransferase Gcn5 and a plasmid to express gRNA recognizing the upstream region of heterochromatic URA3. Confocal microscopy revealed that the fusion proteins were localized in the nucleus. The yeast strain producing the fusion protein and gRNA grew well in the uracil-deficient medium, while the strain harboring empty plasmids or the strain containing the mutations that cause loss of nucleosomal histone acetylation activity of Gcn5 did not. This suggests that the heterochromatin was loosened as much as euchromatin through nucleosomal histone acetylation. The amount of euchromatic DNA at the target locus increased, indicating that chromatin loosening was induced by our system. Nucleosomal histone acetylation in heterochromatic loci by our developed system is a promising method for inducing euchromatic state in a target locus. Full article

Inteins are genetic mobile elements that are inserted within protein-coding genes, which are usually housekeeping genes. They are transcribed and translated along with the host gene, then catalyze their own splicing out of the host protein, which assumes its functional conformation thereafter. As Prp8 inteins are found in several important fungal pathogens and are absent in mammals, they are considered potential therapeutic targets since inhibiting their splicing would selectively block the maturation of fungal proteins. We developed a target-based drug screening system to evaluate the splicing of Prp8 intein from the yeast pathogen Cryptococcus neoformans (CnePrp8i) using Saccharomyces cerevisiae Ura3 as a non-native host protein. In our heterologous system, intein splicing preserved the full functionality of Ura3. To validate the system for drug screening, we examined cisplatin, which has been described as an intein splicing inhibitor. By using our system, new potential protein splicing inhibitors may be identified and used, in the future, as a new class of drugs for mycosis treatment. Our system also greatly facilitates the visualization of CnePrp8i splicing dynamics in vivo. Full article

Kluyveromyces marxianus (K. marxianus) is an increasingly popular industrially relevant yeast. It is known to possess a highly efficient non-homologous end joining (NHEJ) pathway that promotes random integration of non-homologous DNA fragments into its genome. The nature of the integration events was traditionally analyzed by Southern blot hybridization. However, the precise DNA sequence at the insertion sites were not fully explored. We transformed a PCR product of the Saccharomyces cerevisiae URA3 gene (ScURA3) into an uracil auxotroph K. marxianus otherwise wildtype strain and picked 24 stable Ura+ transformants for sequencing analysis. We took advantage of rapid advances in DNA sequencing technologies and developed a method using a combination of Illumina MiSeq and Oxford Nanopore sequencing. This approach enables us to uncover the gross chromosomal rearrangements (GCRs) that are associated with the ScURA3 random integration. Moreover, it will shine a light on understanding DNA repair mechanisms in eukaryotes, which could potentially provide insights for cancer research. Full article

The secretory pathway in Candida albicans involves the protein translocation into the lumen of the endoplasmic reticulum and transport to the Golgi complex, where proteins undergo posttranslational modifications, including glycosylation and proteolysis. The Golgi-resident Kex2 protease is involved in such processing and disruption of its encoding gene affected virulence and dimorphism. These previous studies were performed using cells without URA3 or with URA3 ectopically placed into the KEX2 locus. Since these conditions are known to affect the cellular fitness and the host–fungus interaction, here we generated a kex2Δ null mutant strain with URA3 placed into the neutral locus RPS1. The characterization of this strain showed defects in the cell wall composition, with a reduction in the N-linked mannan content, and the increment in the levels of O-linked mannans, chitin, and β-glucans. The defects in the mannan content are likely linked to changes in Golgi-resident enzymes, as the α-1,2-mannosyltransferase and α-1,6-mannosyltransferase activities were incremented and reduced, respectively. The mutant cells also showed reduced ability to stimulate cytokine production and phagocytosis by human mononuclear cells and macrophages, respectively. Collectively, these data showed that loss of Kex2 affected the cell wall composition, the protein glycosylation pathways, and interaction with innate immune cells. Full article

Fungal keratitis (FK) is a site-threatening infection of the cornea associated with ocular trauma and contact lens wear. Members of the Fusarium solani species complex (FSSC) are predominant agents of FK worldwide, but genes that support their corneal virulence are poorly understood. As a means to bolster genetic analysis in FSSC pathogens, we sought to employ a CRISPR/Cas9 system in an FK isolate identified as Fusarium petroliphilum. Briefly, this approach involves the introduction of two components into fungal protoplasts: (1) A purified Cas9 protein complexed with guide RNAs that will direct the ribonuclease to cut on either side of the gene of interest, and (2) a “repair template” comprised of a hygromycin resistance cassette flanked by 40 bp of homology outside of the Cas9 cuts. In this way, Cas9-induced double strand breaks should potentiate double homologous replacement of the repair template at the desired locus. We targeted a putative ura3 ortholog since its deletion would result in an easily discernable uracil auxotrophy. Indeed, 10% of hygromycin-resistant transformants displayed the auxotrophic phenotype, all of which harbored the expected ura3 gene deletion. By contrast, none of the transformants from the repair template control (i.e., no Cas9) displayed the auxotrophic phenotype, indicating that Cas9 cutting was indeed required to promote homologous integration. Taken together, these data demonstrate that the in vitro Cas9 system is an easy and efficient approach for reverse genetics in FSSC organisms, including clinical isolates, which should enhance virulence research in these important but understudied ocular pathogens. Full article

The essential role of dolichyl phosphate (DolP) as a carbohydrate carrier during protein N-glycosylation is well established. The cellular pool of DolP is derived from de novo synthesis in the dolichol branch of the mevalonate pathway and from recycling of DolPP after each cycle of N-glycosylation, when the oligosaccharide is transferred from the lipid carrier to the protein and DolPP is released and then dephosphorylated. In Saccharomyces cerevisiae, the dephosphorylation of DolPP is known to be catalyzed by the Cwh8p protein. To establish the role of the Cwh8p orthologue in another distantly related yeast species, Candida albicans, we studied its mutant devoid of the CaCWH8 gene. A double Cacwh8∆/Cacwh8∆ strain was constructed by the URA-blaster method. As in S. cerevisiae, the mutant was impaired in DolPP recycling. This defect, however, was accompanied by an elevation of cis-prenyltransferase activity and higher de novo production of dolichols. Despite these compensatory changes, protein glycosylation, cell wall integrity, filamentous growth, and biofilm formation were impaired in the mutant. These results suggest that the defects are not due to the lack of DolP for the protein N-glycosylation but rather that the activity of oligosacharyltransferase could be inhibited by the excess DolPP accumulating in the mutant. Full article

Mutations caused by ion beams have been well-studied in plants, including ornamental flowers, rice, and algae. It has been shown that ion beams have several significantly interesting features, such as a high biological effect and unique mutation spectrum, which is in contrast to low linear energy transfer (LET) radiation such as gamma rays. In this study, the effects of double strand breaks and 8-oxo-2′-deoxyguanosine (8-oxodG) caused by ion-beam irradiation were examined. We irradiated repair-gene-inactive strains rad52, ogg1, and msh2 using carbon ion beams, analyzed the lethality and mutagenicity, and characterized the mutations. High-LET carbon ion-beam radiation was found to cause oxidative base damage, such as 8-oxodG, which can lead to mutations. The present observations suggested that nucleotide incorporation of oxidative damage gave only a limited effect on cell viability and genome fidelity. The ion-beam mutations occurred predominantly in 5′-ACA-3′ sequences, and we detected a hotspot at around +79 to +98 in URA3 in wild-type and mutant lines, suggesting the presence of a mutation-susceptible region. Full article

The NRT2 transporters mediate High Affinity Nitrate/NitriteTransport (HAN/NiT), which are essential for nitrogen acquisition from these inorganic forms. The NRT2 proteins are encoded by a multigene family in plants, and contain 12 transmembrane-spanning domains. Chlamydomonas reinhardtii has six NRT2, two of which (NRT2.5 and NRT2.4) are located in Chromosome III, in tandem head to tail. cDNAs for these genes were isolated and their sequence revealed that they correspond to half-size NRT2 transporters each containing six transmembrane domains. NRT2.5 has long N- and C- termini sequences without known homology. NRT2.4 also contains long termini sequences but smaller than NRT2.5. Expression of both studied genes occurred at a very low level, slightly in darkness, and was not modified by the N or C source. Silencing of NRT2.4 by specific artificial miRNA resulted in the inhibition of nitrite transport in the absence of other HANNiT (NRT2.1/NAR2) in the cell genetic background. Nitrite transport activity in the Hansenula polymorpha Δynt::URA3 Leu2 mutant was restored by expressing CrNRT2.4. These results indicate that half-size NRT2 transporters are present in photosynthetic organisms and that NRT2.4 is a HANiT. Full article

Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER) in Saccharomyces cerevisiae. We have focused on how GGNER relates to histone acetylation for its functioning and we have identified the histone acetyl tranferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. We consider results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then go on to outline the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers throughout entire genomes. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage and then returned to its pre-damaged status to maintain epigenetic codes. Full article