• The Korean Journal of Mycology
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• v.21 no.1
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• pp.9-15
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• 1993

ABSTRACT: The cell fusants were constructed from complementary auxotrophic mutants of Phffia rhodozyma. The nuclear fusion of the fusants was demonstrated by several techniques including comparision of cell volume, estimation of DNA content and nuclear staining. The cell fusants were very stable for succeeding transfer culture on complex medium for more than one year. Malt extract (1%, w/v) and abscisic acid(1 mM) increased the carotenoid formation whereas gibberellic acid(5mM) and riboflavin(0.1 mM) decreased the corresponding content.

• Microbiology and Biotechnology Letters
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• v.27 no.2
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• pp.145-152
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• 1999

Brettanomyces custersii CBS 5512 which has reported as a thermotolerant glucose-cellobiose co-fermentable yeast strain was mutated with UV and NTG to improve ethanol yield at higher than 4$0^{\circ}C$ B. custersii H1-23, H1-39, H1-55 and H1062 were finally selected for hyper-fermentable strains at higher than 4$0^{\circ}C$ from thermotolerant 7510 colonies through 5th selection. Among the selected strains, H1-39 mutant had better fermentability at 4$0^{\circ}C$ and 43$^{\circ}C$ from different concentrations of glucose. H1-39 and H1-23 mutants yielded more than 70% of the theoretical ethanol yield in 4 and 8% mixed sugars at above 4$0^{\circ}C$, which was 5-11% higher than those by original strain. Especially, H1-39 mutant had better fermentability in 4% mixed sugar. It showed 78.5% of the theoretical yield at 4$0^{\circ}C$ and 72.2% of the theoretical yield at 43$^{\circ}C$. On the other hand, theoretical yield of ethanol by H1-39 mutant in 8% mixed sugar at 4$0^{\circ}C$ and 43$^{\circ}C$ were 75.2% and 70.2%, respectively. Theses values increased up to 7-11% as compared to those by orginal strain. By the simultaneous saccharification and fermentation, ethanol production by H1-39 mutant increased up to more than 23% as compared to that by original strain.

• The Plant Pathology Journal
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• v.19 no.1
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• pp.24-29
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• 2003

SS2-2, a hypernodulating soybean mutant was isolated by EMS mutagenesis from Sinpaldalkong 2. This auto-regulation mutant showed greater number of nodules and smaller plant size than its wild type Sinpaldalkong 2. SSR markers were used to identify DNA variation at SSR loci from different soybean LG. The only SSR marker that detected a length polymorphism between SS2-2 and its wild type ancestor was Satt294 on LG C1 instead of LG H, locating a hypernodulating gene. Sequencing data of flanking Satt294 indicated that the size variation was due to extra stretch of TTA repeats of the SSR motif in SS2-2, along with $A\longrightarrow$G transversion. In spite of phenotypic differences between the wild type and its hypernodulating mutants, genomic DNA poly-morphisms at microsatellite loci could not control regulation of nodule formation. The cDNA-AFLP method was applied to compare differential display of cDNA between Sinpaldalkong 2 and SS2-2. After isolation and sequence comparison with many AELP fragments, several interesting genes were identified. Northern blot analysis, immunolocalization and/or the yeast two-hybrid system with these genes might provide information on regulation of nodule development in SS2-2.

• Microbiology and Biotechnology Letters
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• v.19 no.5
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• pp.491-496
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• 1991

To maximize the production of aminoglycoside-3'-phosphotransferase of E. coli ATCC 21990 carrying R factor which encodes aminoglycoside-3'-phosphotransferase (APH(3')) phosphorylating the 3'-hydroxyl group of aminoglycoside, mutants M1 and M2, media composition and several factors affecting the enzyme production during fermentation were studied. Although the specific activity of APH(3') produced by a mutant M1 was increased as much as four times than that of E. coii ATCC 21990, the growth rate was decreased. The increase of the enzyme production was obtained by increased biomass during fermentation. A mutant M2 was obtained to increase the cell growth rate. Mutant M2 cells were cultivated with optimal media and pure oxygen gas in a fed-batch mode of fermentor operation. The specific activity of APH(3') was decreased, but total enzyme activity of APH(3') was increased as much as two point five times than that of mutant MI.

• Molecules and Cells
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• v.38 no.12
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• pp.1054-1063
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• 2015

Mitochondria play a crucial role in eukaryotic cells; the mitochondrial electron transport chain (ETC) generates adenosine triphosphate (ATP), which serves as an energy source for numerous critical cellular activities. However, the ETC also generates deleterious reactive oxygen species (ROS) as a natural byproduct of oxidative phosphorylation. ROS are considered the major cause of aging because they damage proteins, lipids, and DNA by oxidation. We analyzed the chronological life span, growth phenotype, mitochondrial membrane potential (MMP), and intracellular ATP and mitochondrial superoxide levels of 33 single ETC component-deleted strains during the chronological aging process. Among the ETC mutant strains, 14 ($sdh1{\Delta}$, $sdh2{\Delta}$, $sdh4{\Delta}$, $cor1{\Delta}$, $cyt1{\Delta}$, $qcr7{\Delta}$, $qcr8{\Delta}$, $rip1{\Delta}$, $cox6{\Delta}$, $cox7{\Delta}$, $cox9{\Delta}$, $atp4{\Delta}$, $atp7{\Delta}$, and $atp17{\Delta}$) showed a significantly shorter life span. The deleted genes encode important elements of the ETC components succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV), and some of the deletions lead to structural instability of the membrane-$F_1F_0$-ATP synthase due to mutations in the stator stalk (complex V). These short-lived strains generated higher superoxide levels and produced lower ATP levels without alteration of MMP. In summary, ETC mutations decreased the life span of yeast due to impaired mitochondrial efficiency.

• Bulletin of the Korean Chemical Society
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• v.22 no.5
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• pp.514-518
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• 2001

We have explored the importance of two ATP binding domains of Hsp104 protein in protection of yeast cells from cadmium exposure. In the previous study we have discovered that the presence of two ATP binding sites was essential in providing heat sh ock protection as well as rescuing cells from oxidative stress. In this paper we first report wild type cell with functional hsp104 gene is more resistant to cadmium stress than hsp104-deleted mutant cell, judging from decrease in survival rates as a result of cadmium exposure. In order to demonstrate functional role of two ATP binding sites in cadmium defense, we have transformed both wild type (SP1) and hyperactivated ras mutant (IR2.5) strains with several plasmids differing in the presence of ATP binding sites. When an extra copy of functional hsp104 gene with both ATP binding sites was overexpressed with GPD-promoter, cells showed increased survival rate against cadmium stress than mutants with ATP binding sites changed. The degree of protection in the presence of two ATP binding sites was similarly observed in ira2-deleted hyperactivated ras mutant, which was more sensitive to oxidative stress than wild type cell. We have concluded that the greater sensitivity to cadmium stress in the absence of two ATP binding sites is attributed to the higher concentration of reactive oxygen species (ROS) produced by cadmium exposure based on the fluorescence tests. These findings, taken all together, imply that the mechanism by which cadmium put forth toxic effects may be closely associated with the oxidative stress, which is regulated independently of the Ras-cAMP pathway. Our study provides a better understanding of cadmium defense itself and cross-talks between oxidative stress and metal stress, which can be applied to control human diseases due to similar toxic environments.

• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.79-91
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• 2021

γ-Glutamylcysteine synthetase (Gcs1) and glutathione reductase (Glr1) activity maintains minimal levels of cellular methylglyoxal in Candida albicans. In glutathione-depleted Δgcs1, we previously saw that NAD(H)-linked methylglyoxal oxidoreductase (Mgd1) and alcohol dehydrogenase (Adh1) are the most active methylglyoxal scavengers. With methylglyoxal accumulation, disruptants lacking MGD1 or ADH1 exhibit a poor redox state. However, there is little convincing evidence for a reciprocal relationship between methylglyoxal scavenger genes-disrupted mutants and changes in glutathione-(in)dependent redox regulation. Herein, we attempt to demonstrate a functional role for methylglyoxal scavengers, modeled on a triple disruptant (Δmgd1/Δadh1/Δgcs1), to link between antioxidative enzyme activities and their metabolites in glutathione-depleted conditions. Despite seeing elevated methylglyoxal in all of the disruptants, the result saw a decrease in pyruvate content in Δmgd1/Δadh1/Δgcs1 which was not observed in double gene-disrupted strains such as Δmgd1/Δgcs1 and Δadh1/Δgcs1. Interestingly, Δmgd1/Δadh1/Δgcs1 exhibited a significantly decrease in H2O2 and superoxide which was also unobserved in Δmgd1/Δgcs1 and Δadh1/Δgcs1. The activities of the antioxidative enzymes erythroascorbate peroxidase and cytochrome c peroxidase were noticeably higher in Δmgd1/Δadh1/Δgcs1 than in the other disruptants. Meanwhile, Glr1 activity severely diminished in Δmgd1/Δadh1/Δgcs1. Monitoring complementary gene transcripts between double gene-disrupted Δmgd1/Δgcs1 and Δadh1/Δgcs1 supported the concept of an unbalanced redox state independent of the Glr1 activity for Δmgd1/Δadh1/Δgcs1. Our data demonstrate the reciprocal use of Eapx1 and Ccp1 in the absence of both methylglyoxal scavengers; that being pivotal for viability in non-filamentous budding yeast.

• Development and Reproduction
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• v.12 no.3
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• pp.289-296
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• 2008

BPES (Blepharophimosis/Ptosis/Epicanthus inversus Syndrome) is an autosomal dominant disorder caused by mutations in FOXL2. Affected individuals have premature ovarian failure (POF) in addition to small palpebral fissures, drooping eyelids, and broad nasal bridge. FOXL2 is a member of the forkhead family transcription factors. In FOXL2-deficient ovaries, granulosa cell differentiation dose not progress, leading to arrest of folliculogenesis and oocytes atresia. Using yeast two-hybrid screening of rat ovarian cDNA library with FOXL2 as bait, we found that small ubiquitin-related modifier (SUMO)-conjugating E2 enzyme UBE2I protein interacted with FOXL2 protein. UBE2I also known as UBC9 is an essential protein for processing SUMO modification. Sumoylation is a form of post-translational modification involved in diverse signaling pathways including the regulation of transcriptional activities of many transcriptional factors. In the present study, we confirmed the protein-protein interaction between FOXL2 and UBE2I in human cells, 293T, by in vivo immunoprecipitation. In addition, we generated truncated FOXL2 mutants and identified the region of FOXL2 required for its association with UBE2I using yeast-two hybrid system. Therefore, the identification of UBE2I as an interacting protein of FOXL2 further suggests a presence of novel regulatory mechanism of FOXL2 by sumoylation.

• KSBB Journal
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• v.22 no.5
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• pp.305-312
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• 2007

It is crucial to develop a miniaturized cultivation method for large and rapid screening of high-yielding mutants of monacolin-K, a powerful anti-hypercholesterolemic secondary metabolite biosynthesized by the fungal cells of Monascus ruber. In order to investigate as many strains as possible in a short time, a miniaturized fermentation method especially suitable for the cultivation of the filamentous Monascus mutants was developed using $50m{\ell}$ culture-tube ($7m{\ell}$ of working volume) instead of the traditional $250m{\ell}$ flask ($50m{\ell}$ of working volume). Generally, in filamentous fungal cell fermentations, morphologies in growth and production cultures should be maintained as thick filamentous and compact-pelleted (usually less than 1 mm in diameter) forms, respectively, for enhanced production of secondary metabolites in final production cultures. In this study, we intended to induce the respective optimal morphologies in the miniaturized culture system for the purpose of rapid screening of overproducers. Miniaturized growth culture system was successfully developed due to the mass production of spores in the statistically optimized solid medium. When large amounts of spores were inoculated into the growth cultures, and brown rice flour (20 g/L) was also supplemented to the growth medium, dense filamentous morphologies were successfully induced in the growth cultures performed with the 50 ml culture tubes. It was implied that the amounts of spores inoculated into the growth tube-cultures and the growth medium components should be the key factors for the induction of the filamentous forms in the growth fermentations. Furthermore, in order to statistically optimize production medium, multiple experiments based on Plackett-Burman design and response surface method (RSM) were carried out, resulting in more than 2 fold enhanced production of monacolin-K in the final production cultures with the optimized production medium. Notably, under the production culture conditions with the statistically optimized medium, optimal pellet sizes below 1 mm in diameter were reproducibly induced, in contrast to the thick and viscous filamentous morphologies observed in the previous production cultures.

• Korean Journal of Food Science and Technology
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• v.31 no.2
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• pp.465-474
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• 1999

RNase activity of Saccharomyces cerevisiae ATCC 7754 was investigated to obtain strains with high ribonucleic acid (RNA) content. The yeast strain contained two RNase activities; an acidic RNase with a optima of pH $3{\sim}4$ and an alkaline RNase with a optima pH 9. The acidic RNase activity was inhibited by $0.08\;M\;HgCl_{2}$ most drastically. The alkaline RNase activity was inhibited by 2.0 M NaCl or KCl, while enhanced by addition of $0.05\;M\;CaCl_{2},\;0.02\;M\;ZnSO_{4},\;or\;0.008\;M\;HgCl_{2}$. Various mutants of Saccharomyces cerevisiae ATCC 7754 were isolated by ethylmethane sulfonate (EMS) treatment or $\gamma$-ray/ultra violet irradiation. Among the mutants that were sensitive to high concentration of KCl which inhibits alkaline RNase, B24 was selected for high RNA content per culture volume. Growth characteristics of the mutant were comparable to those of the mother strain with optimum growth at pH $4.5{\sim}5.5$. The mutant accumulated higher content of RNA than the mother strain when glucose was used as the carbon source. However, both growth rate and total RNA content of the mutant were higher in molasses medium than in glucose medium. RNA content of the mutant increased rapidly during the early stage of growth, and then decreased gradually until the culture reached stationary phase by a fed-batch culture in a 5 L jar fermenter. Maximal cell harvest and the final RNA content using the mutant B24 were 69.6 g/L culture broth and 19.8 g/100 g of the dry cell while those using the mother strain were 68 g/L culture broth and 16.1 g/100 g of dry cell, respectively.