Myoung-Dong Kim

A novel F-box protein involved in sexual development and pathogenesis in Gibberella zeae

Gibberella zeae is an ascomyceteous fungus that causes serious diseases in cereal crops. Severe epidemics require strains that are virulent and that can reproduce sexually. We characterized an insertional mutant (designated ZH436) with a pleiotropic defect in both traits, and identified a novel F‐box protein gene encoding FBP1 (F‐box protein 1) that is similar to fungal F‐box proteins including Saccharomyces cerevisiae Grr1, a well‐characterized component of the Skp1‐Cullin‐F‐box protein (SCFGrr1) E3 ligase complex required for protein degradation. FBP1 also can bind both S. cerevisiae Skp1 protein, the other component of the SCFGrr1 complex, and its G. zeae sequence homologue SKP1. Two putative protein interacting domains in FBP1 are essential for in vivo function. FBP1 and ScGRR1 are not so interchangeable between S. cerevisiae and G. zeae, but FBP1 can partially complement several defects of a yeast grr1 deletion mutant. Functional analyses confirmed that FBP1 is required for several phenotypes including both sexual development and virulence in G. zeae; the phenotype of ΔFBP1 strains is different from those of null mutants for F‐box proteins in other filamentous fungi as well as from S. cerevisiae grr1Δ strains. Thus, FBP1 is a versatile F‐box protein that presumably participates in the formation of the SCFFBP1 complex that probably controls the ubiquitin‐mediated degradation of proteins involved in sexual reproduction and virulence important for disease development by G. zeae.

GzSNF1 is required for normal sexual and asexual development in the ascomycete Gibberella zeae.

ABSTRACT The sucrose nonfermenting 1 (SNF1) protein kinase of yeast plays a central role in the transcription of glucose-repressible genes in response to glucose starvation. In this study, we deleted an ortholog of SNF1 from Gibberella zeae to characterize its functions by using a gene replacement strategy. The mycelial growth of deletion mutants (ΔGzSNF1) was reduced by 21 to 74% on diverse carbon sources. The virulence of ΔGzSNF1 mutants on barley decreased, and the expression of genes encoding cell-wall-degrading enzymes was reduced. The most distinct phenotypic changes were in sexual and asexual development. ΔGzSNF1 mutants produced 30% fewer perithecia, which matured more slowly, and asci that contained one to eight abnormally shaped ascospores. Mutants in which only the GzSNF1 catalytic domain was deleted had the same phenotype changes as the ΔGzSNF1 strains, but the phenotype was less extreme in the mutants with the regulatory domain deleted. In outcrosses between the ΔGzSNF1 mutants, each perithecium contained ∼70% of the abnormal ascospores, and ∼50% of the asci showed unexpected segregation patterns in a single locus tested. The asexual spores of the ΔGzSNF1 mutants were shorter and had fewer septa than those of the wild-type strain. The germination and nucleation of both ascospores and conidia were delayed in ΔGzSNF1 mutants in comparison with those of the wild-type strain. GzSNF1 expression and localization depended on the developmental stage of the fungus. These results suggest that GzSNF1 is critical for normal sexual and asexual development in addition to virulence and the utilization of alternative carbon sources.

Production of resveratrol from p-coumaric acid in recombinant Saccharomyces cerevisiae expressing 4-coumarate:coenzyme A ligase and stilbene synthase genes.

Resveratrol is a well-known polyphenol present in red wine and exerts antioxidative and anti-carcinogenic effects on the human body. To produce resveratrol in a food-grade yeast, the 4-coumarate:coenzyme A ligase gene (4CL1) from Arabidopsis thaliana and stilbene synthase gene (STS) from Arachis hypogaea were cloned and transformed into Saccharomyces cerevisiae W303-1A. The resveratrol produced was unglycosylated and secreted into the culture medium. A batch culture with 15.3mg/l p-coumaric acid used as precursor resulted in the production of 3.1mg/l resveratrol with 14.4 mol% yield. Deletion of the putative phenyl acrylic acid decarboxylase gene (PAD1) did not enhance resveratrol production.

Production of resveratrol from tyrosine in metabolically engineered Saccharomyces cerevisiae.

Resveratrol, a polyphenol compound found in grape skins, has been proposed to account for the beneficial effects of red wine against heart disease. To produce resveratrol in Saccharomyces cerevisiae, four heterologous genes were introduced: the phenylalanine ammonia lyase gene from Rhodosporidium toruloides, the cinnamic acid 4-hydroxylase and 4-coumarate:coenzyme A ligase genes both from Arabidopsis thaliana, and the stilbene synthase gene from Arachis hypogaea. When this recombinant yeast was cultivated by batch fermentation in YP medium containing 2% galactose, it produced 2.6 mg/L p-coumaric acid and 3.3 mg/L resveratrol. In order to increase the pool of malonyl-CoA, a key precursor in resveratrol biosynthesis, the acetyl-CoA carboxylase (ACC1) gene was additionally overexpressed in the yeast by replacing the native promoter of the ACC1 gene with the stronger GAL1 promoter and this resulted in enhanced production of resveratrol (4.3 mg/L). Furthermore, when tyrosine was supplemented in the medium, the concentration of resveratrol increased up to 5.8 mg/L. This result illustrates a possible strategy for developing metabolically engineered yeast strain for the economical production of resveratrol from cheap amino acids.

Monitoring of Leuconostoc mesenteroides DRC starter in fermented vegetable by random integration of chloramphenicol acetyltransferase gene

In 2004, Leuconostoc mesenteroides DRC was first used as a starter culture for achieving higher organoleptic effects in Korean kimchi manufacture. For a better understanding of starter growth in a mixed culture system, and for predicting starter predominance in kimchi, a monitoring system for the starter was established. The chloramphenicol resistance marker gene (cat) was randomly integrated into chromosomal DNA of L. mesenteroides DRC using a viral transposon and transposase. The DRC mutant, tDRC2, had a similar growth pattern to the host strain, with no major alteration in phenotypic characteristics. The mutant strain was inoculated into real kimchi, and monitoring of the starter population was successfully achieved. The overall predominance of Leuconostoc in kimchi inoculated with DRC followed the general growth pattern of this genus during kimchi fermentation. Our results also demonstrate the competitive ability of the DRC starter against Leuconostoc from natural flora, maintaining its predominance above 88% during the whole fermentation period. Based on this experiment, the random gene integration method using a transposon was shown to be of utility in transferring any commercial starter into a selectable and monitorable strain for simulation purposes.

Sepiapterin inhibits cell proliferation and migration of ovarian cancer cells via down-regulation of p70S6K-dependent VEGFR-2 expression

Tetrahydrobiopterin (BH4) is known to be an essential cofactor for the aromatic amino acid hydroxylases, which are involved in the production of neurotransmitters, and for nitric oxide (NO) synthase. In the present study, we report that sepiapterin, the more stable form of the BH4 precursor, modulates ovarian cancer cell proliferation and migration by NO-dependent and -independent mechanisms. Sepiapterin induction of cell proliferation and migration in SKOV-3 cells is accompanied by ERK, Akt and p70S6K activation. These stimulatory effects of sepiapterin are reversed by pretreatment with NO synthase inhibitor. We also show that sepiapterin significantly inhibits vascular endothelial growth factor-A (VEGF-A)-stimulated cell proliferation and migration. Pretreatment with NO synthase inhibitor does not alter the ability of sepiapterin to inhibit VEGF-A-induced cell proliferation and migration, indicating that the suppressive effects of sepiapterin on VEGF-A-induced responses are mediated by a NO-independent mechanism. Finally, we demonstrate that sepiapterin markedly suppresses VEGF-A-induced p70S6K phosphorylation and VEGFR-2 expression, resulting in inhibition of VEGF-A-induced cell proliferation and migration. Collectively, these findings represent a biphasic effect of sepiapterin on cellular fates, depending on the presence of growth factors, and support further development and evaluation of sepiapterin for the treatment of cancers overexpressing VEGFR-2.

Cloning and characterization of CmGPD1, the Candida magnoliae homologue of glycerol‐3‐phosphate dehydrogenase

Glycerol-3-phosphate dehydrogenase (GPDH) plays a central role in glycerol metabolism. A genomic CmGPD1 gene encoding NADH-dependent GPDH was isolated from Candida magnoliae producing a significant amount of glycerol. The gene encodes a polypeptide of 360 amino acids, which shows high homology with known NADH-dependent GPDHs of other species. The CmGPD1 gene was expressed in recombinant Escherichia coli with the maltose-binding protein (MBP) fusion system and purified to homogeneity using simple affinity chromatography. The purified CmGpd1p without the MBP fusion displayed an apparent molecular mass of 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The CmGpd1p enzyme exhibited a K(cat)/K(m) value of 195 min(-1) mM(-1) for dihydroxyacetone phosphate whereas K(cat)/K(m) for glycerol-3-phosphate is 0.385 min(-1) mM(-1). In a complementation study, CmGpd1p rescued the ability of glycerol synthesis and salt tolerance in a Saccharomyces cerevisiae GPD1DeltaGPD2Delta mutant strain. The overall results indicated that CmGPD1 encodes a functional homologue of S. cerevisiae GPDH.

Inhibitory effects of sepiapterin on vascular endothelial growth factor-a-induced proliferation and adhesion in human umbilical vein endothelial cells

Tetrahydrobiopterin (BH4) has been known to be an essential cofactor for the activities of nitric oxide (NO) synthase and aromatic amino acid hydroxylases, which are involved in physiological and pathological processes. In the present study, we report that sepiapterin, the more stable form of BH4 precursor, modulates vascular endothelial growth factor-A (VEGF-A)-induced cell proliferation and adhesion in human umbilical vein endothelial cells (HUVECs). The antiproliferative activity of sepiapterin in VEGF-A-treated HUVECs is associated with inhibition of the expression of cyclin-dependent kinases (Cdks) such as Cdk4 and Cdk2. Pretreatment with NO synthase inhibitor does not abrogate the ability of sepiapterin to inhibit VEGF-A-induced cell proliferation and adhesion, indicating that the suppressive effects of sepiapterin on VEGF-Ainduced responses are mediated by NO-independent mechanism. Finally, we show that sepiapterin modulates VEGF-A-induced cell proliferation and adhesion through down-regulation of VEGF receptor-2 downstream signaling pathways. Taken together, these findings represent a novel function of sepiapterin in the regulation of angiogenesis, supporting further development and evaluation of sepiapterin as an antiangiogenic agent.

Cysteine Protease Profiles of the Medicinal Plant Calotropis procera R. Br. Revealed by De Novo Transcriptome Analysis

Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding.

Nuruk, a traditional korean fermentation starter, contains the bioactive compound 2,6-dimethoxy-1,4-benzoquinone (2,6-DMBQ)

Nuruk, a traditional Korean fermentation starter, contains 2,6-dimethoxy-ρ-benzoquinoe (2,6-DMBQ), also found in fermented wheat germ extract, and has anti-cancer and immune supporting effects. The presence of 2,6-DMBQ was confirmed by high performance liquid chromatography and mass spectrometry. Among the five traditional nuruks tested, the highest 2,6-DMBQ content of 1.16±0.07 mg/50 g was obtained from nuruk purchased in Hwaseong. The results of this study may explain the health-promoting functions of traditional Korean alcoholic beverages that employ nuruk as a fermentation starter.