Dae-Hyuk Kim

Characterization of HOG1 homologue, CpMK1, from Cryphonectria parasitica and evidence for hypovirus-mediated perturbation of its phosphorylation in response to hypertonic stress

We examined the biological function of cpmk1, which encodes a MAPK of Cryphonectria parasitica, and its regulation by mycovirus. Sequence comparisons revealed that cpmk1 had highest homology with osm1, a hog1‐homologue from Magnaporthe grisea. A growth defect was observed in the cpmk1‐null mutant under hyperosmotic conditions, indicating that cpmk1 functionally belongs to a hog1 subfamily. Immunoblot analyses indicated that the CpMK1 pathway was affected specifically in hyperosmotic conditions by the hypovirus CHV1‐EP713. Moreover, the virus‐infected hypovirulent UEP1 strain also exhibited severe osmosensitivity compared to the virus‐free isogenic strain EP155/2, thus providing additional evidence for viral regulation of cpmk1 in response to a hypertonic stress. Besides osmosensitivity, disruption of cpmk1 resulted in several, but not all, hypovirulence‐associated changes, such as reduced pigmentation, conidiation, laccase production and cryparin expression. However, the cpmk1‐null mutant exhibited an increased accumulation of pheromone gene transcripts. Virulence assays of the cpmk1‐null mutant revealed reduced canker area, but not as severe as that of UEP1. These results suggest that mycoviruses modulate the MAPK and thereby provoke the aberrant expression of target genes, some of which are likely to be implicated in viral symptom development.

Expression of glucose oxidase by using recombinant yeast

The glucose oxidase gene (GO) of Aspergillus niger was cloned into the yeast shuttle vector YEp352 with combinations of various promoters and terminators, and then used to transform Saccharomyces cerevisiae. Expressed GO was successfully secreted into culture medium due to the presence of the intrinsic signal peptide of GO. Four different promoters fused to GO were tested: bidirectional galactose dehydrogenase 1 and 10 (GAL1, GAL10) promoters, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and an yeast hybrid ADH2-GPD promoter consisting of alcohol dehydrogenase II (ADH2) and GPD promoter. The intrinsic terminator of GO as well as the GAL7 terminator were also compared for better production of GO. Deletion of most of the terminating region from GO yielded only a slight amount of GO while the presence of either terminator greatly increased GO production. The GAL10 promoter produced the least amount of GO, GAL1 and GPD promoters were moderate, and the ADH2-GPD hybrid promoter was the best among all tested. However, the hybrid promoter was tightly regulated by the presence of an excess amount of either glucose or ethanol, and it appeared that 2% glucose and 1. 5% ethanol supplement was the best concentration for GO production. It was possible to produce 260 IU ml(-1) of GO, an equivalent of 5 g l(-1), under the presence of 2% glucose and 1.5% ethanol. UV mutagenesis of a recombinant S. cerevisiae was also applied and it further increased the yield of GO to 460 IU ml(-1) under the presence of 2% glucose and 1.5% ethanol without any changes in cell growth. Corn steep liquor which is commonly used in bioindustry is a good alternative substrate for high priced glucose for the hybrid promoter and suggests a cost effective means for commercial mass production of GO using recombinant yeast.

Improved ethanol tolerance in Escherichia coli by changing the cellular fatty acids composition through genetic manipulation

To investigate the effect of cellular fatty acids composition on ethanol tolerance in Escherichia coli, we overexpressed either des, encoding fatty acid desaturase from Bacillus subtilis, or fabA, encoding β-hydroxydecanoyl thio-ester dehydrase from E. coli, or both genes together, into E. coli. Recombinant E. coli harboring fabA had elevated tolerance against ethanol compared to wild type strain. In contrast, des decreased resistance to ethanol. Co-expression of both genes together complemented ethanol tolerance of E. coli. This result indicates how to engineer bacterial strains to be resistant to higher concentrations of ethanol.

Characterization of a fungal protein kinase from Cryphonectria parasitica and its transcriptional upregulation by hypovirus

The chestnut blight fungus Cryphonectria parasitica and its hypovirus comprise useful model system to study the mechanisms of hypoviral infection. We used degenerate primers based on fungal protein kinases to isolate a gene, cppk1, which encodes a novel Ser/Thr protein kinase of C. parasitica. The gene showed highest homology to ptk1, a Ser/Thr protein kinase from Trichoderma reesei. The encoded protein had a predicted mass of 70.5 kDa and a pI of 7.45. Northern blot analyses revealed that the cppk1 transcript was expressed from the beginning of culture, with a slight increase by 5 days of culture. However, its expression was specifically affected by the presence of virus, and it was transcriptionally upregulated in the fungal strain infected with the hypovirus. A kinase assay using Escherichia coli‐derived CpPK1 revealed CpPK1‐specific phosphorylated proteins with estimated masses of 50 kDa and 44 kDa. In addition, the phosphorylation of both proteins was higher in a cell‐free extract from the hypovirulent strain. The increased expression of cppk1 by the introduction of an additional copy results in a subset of viral symptoms of reduced pigmentation and conidiation in a virus‐free isolate. cppk1 overexpression also causes the downregulation of mating factor genes Mf2/1 and Mf2/2, resulting in female sterility. The present study suggests that the hypovirus disturbs fungal signalling by transcriptional upregulation of cppk1, which results in reduced pigmentation and conidiation and female sterility.

Expression of Heteropolymeric Ferritin Improves Iron Storage in Saccharomyces cerevisiae

ABSTRACT Saccharomyces cerevisiae was engineered to express different amount of heavy (H)- and light (L)-chain subunits of human ferritin by using a low-copy integrative vector (YIp) and a high-copy episomal vector (YEp). In addition to pep4::HIS3 allele, the expression host strain was bred to have the selection markers leu2− and ura3− for YIplac128 and YEp352, respectively. The heterologous expression of phytase was used to determine the expression capability of the host strain. Expression in the new host strain (2805-a7) was as high as that in the parental strain (2805), which expresses high levels of several foreign genes. Following transformation, Northern and Western blot analyses demonstrated the expression of H- and L-chain genes. The recombinant yeast was more iron tolerant, in that transformed cells formed colonies on plates containing more than 25 mM ferric citrate, whereas none of the recipient strain cells did. Prussian blue staining indicated that the expressed isoferritins were assembled in vivo into a complex that bound iron. The expressed subunits showed a clear preference for the formation of heteropolymers over homopolymers. The molar ratio of H to L chains was estimated to be 1:6.8. The gel-purified heteropolymer took up iron faster than the L homopolymer, and it took up more iron than the H homopolymer did. The iron concentrations in transformants expressing the heteropolymer, L homopolymer, and H homopolymer were 1,004, 760, and 500 μg per g (dry weight) of recombinant yeast cells, respectively. The results indicate that heterologously expressed H and L subunits coassemble into a heteropolymer in vivo and that the iron-carrying capacity of yeast is further enhanced by the expression of heteropolymeric isoferritin.

Enhanced iron uptake of Saccharomyces cerevisiae by heterologous expression of a tadpole ferritin gene.

ABSTRACT We genetically engineered Saccharomyces cerevisiae to express ferritin, a ubiquitous iron storage protein, with the major heavy-chain subunit of tadpole ferritin. A 450-kDa ferritin complex can store up to 4,500 iron atoms in its central cavity. We cloned the tadpole ferritin heavy-chain gene (TFH) into the yeast shuttle vector YEp352 under the control of a hybrid alcohol dehydrogenase II and glyceraldehyde-3-phosphate dehydrogenase promoter. We confirmed transformation and expression by Northern blot analysis of the recombinant yeast, by Western blot analysis using an antibody against Escherichia coli-expressed TFH, and with Prussian blue staining that indicated that the yeast-expressed tadpole ferritin was assembled into a complex that could bind iron. The recombinant yeast was more iron tolerant in that 95% of transformed cells, but none of the recipient strain cells, could form colonies on plates containing 30 mM ferric citrate. The cell-associated concentration of iron was 500 μg per gram (dry cell weight) of the recombinant yeast but was 210 μg per gram (dry cell weight) in the wild type. These findings indicate that the iron-carrying capacity of yeast is improved by heterologous expression of tadpole ferritin and suggests that this approach may help relieve dietary iron deficiencies in domesticated animals by the use of the engineered yeast as a feed and food supplement.

A Tannic Acid-Inducible and Hypoviral-Regulated Laccase3 Contributes to the Virulence of the Chestnut Blight Fungus Cryphonectria parasitica

A new laccase gene (lac3) from the chestnut blight fungus Cryphonectria parasitica was induced by the presence of tannic acid, which is abundant in the bark of chestnut trees and is assumed to be one of the major barriers against pathogen infection. However, other commonly known laccase inducers, including ferulic acid, 2,5-xylidine, catechol, and pH, did not induce lac3 transcription. Moreover, the hypovirus modulated the induction of lac3 transcription, abolishing the transcriptional induction of the lac3 gene by tannic acid. A functional analysis of lac3 using a lac3-null mutant indicated that fungal growth and other morphological characteristics, including pigmentation and sporulation, were not affected. However, a virulence assay indicated that the loss of function of a tannic acid-inducible and hypoviral-regulated laccase resulted in reduced virulence without detectable changes in the morphological features. The constitutive expression of lac3 resulted in no significant differences in the necrotic lesions from those caused by the wild type, but its expression in the presence of the hypovirus led to larger lesions than those caused by the hypovirulent strain. These results suggest that the lac3 gene product may not be the only determinant of fungal virulence in chestnut trees but is an important factor.

Surface-Displayed Expression of a Neutralizing Epitope of ApxIIA Exotoxin in Saccharomyces cerevisiae and Oral Administration of It for Protective Immune Responses against Challenge by Actinobacillus pleuropneumoniae

A neutralizing epitope fragment of ApxIIA toxin (ApxIIA#5) of the Korean Actinobacillus pleuropneumoniae serotype 2 strain was expressed and immobilized on the cell surface of Saccharomyces cerevisiae for efficient vaccine development. Expression of ApxIIA#5 was confirmed by Western blot analysis using cell-wall proteins, and the surface display of ApxIIA#5 was further visualized under confocal microscopy. Quantitative ELISA revealed that the recombinant ApxIIA#5 directed to the cell surface consisted of approximately 16% cell-wall proteins, estimated to be 35 mg of ApxIIA#5 protein per liter of cultured cells. An immunoassay revealed that antigen-specific antibodies against ApxIIA#5 were present in the sera of mice fed recombinant ApxIIA#5-expressing yeast, but not in mice fed the wild-type nor the vector-only transformant. Moreover, the mice fed the recombinant epitope-expressing yeast were protected from injection of a lethal dose of A. pleuropneumoniae.

Heterologous expression and characterization of class III chitinases from rice (Oryza sativa L.)

Abstract Acidic (OsChib1a) and basic (OsChib1b) class III chitinases from rice ( Oryza sativa L.), sharing 70% of the identical amino acid residues each other, were expressed from the corresponding cDNAs in Pichia pastoris and purified homogenously. Both OsChib1a and OsChib1b degraded actively glycol chitin over colloidal chitin at the optimum pH of 4.3 and 8.3, respectively. OsChib1b had lower specific chitinase activity than OsChib1a, but it showed a strong lytic activity and significant antifungal activity: no lytic and antifungal activity was observed for OsChib1a. Experiments with N -acetyl chitooligosaccharides showed that OsChib1a hydrolyzed (GlcNAc) 6 efficiently to yield (GlcNAc) 2 as the major product, while OsChib1b hydrolyzed (GlcNAc) 6 to yield both (GlcNAc) 2 and (GlcNAc) 4 . Possible structures responsible for their distinct catalytic properties are discussed.

Transformation of a filamentous fungusCryphonectria parasitica usingAgrobacterium tumefaciens

AsAgrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast,Saccharomyces cerevisiae, a variety of fungi were subjected to theA. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. TheA. tumefaciens-mediated transformation of chestnut blight fungus,Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of theAspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1×106 conidia ofC. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.