• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.8-15
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• 2021

More and more available fungal genome sequence data reveal a large amount of secondary metabolite (SM) biosynthetic 'dark matter' to be discovered. Heterogeneous expression is one of the most effective approaches to exploit these novel natural products, but it is limited by having to clone entire biosynthetic gene clusters (BGCs) without errors. So far, few effective technologies have been developed to manipulate the specific large DNA fragments in filamentous fungi. Here, we developed a fungal BGC-capturing system based on CRISPR/Cas9 cleavage in vitro. In our system, Cas9 protein was purified and CRISPR guide sequences in combination with in vivo yeast assembly were rationally designed. Using targeted cleavages of plasmid DNAs with linear (8.5 kb) or circular (8.5 kb and 28 kb) states, we were able to cleave the plasmids precisely, demonstrating the high efficiency of this system. Furthermore, we successfully captured the entire Nrc gene cluster from the genomic DNA of Neosartorya fischeri. Our results provide an easy and efficient approach to manipulate fungal genomic DNA based on the in vitro application of Cas9 endonuclease. Our methodology will lay a foundation for capturing entire groups of BGCs in filamentous fungi and accelerate fungal SMs mining.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2002.04b
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• pp.19-25
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• 2002

Biotechnology in the 21st century will be driven by three emerging technologies: genomics, high-throughput biology, and bioinformatics. These technologies are complementary to one another. A large number of economically important crops are currently subjected to whole genome sequencing. Functional genomics for determining the functions of the genes comprising the given plant genome is under progress by using various means including phenotyping data from transgenic mutants, gene expression profiling data from DNA microarrays, and metabolic profiling data from LC/mass analysis. The aim of plant molecular breeding is shifting from introducing agronomic traits such as herbicide and insect resistance to introducing quality traits such as healthful oils and proteins, which will lead to improved and nutritional food and feed products. Plant molecular breeding is also expected to aim to develop crops for producing human therapeutic and industrial proteins.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2000.11a
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• pp.13-16
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• 2000

Microorganisms have been widely employed for the production of useful bioproducts including primary metabolites such as ethanol, succinic acid, acetone and butanol, secondary metabolites represented by antibiotics, proteins, polysaccharides, lipids and many others. Since these products can be obtained in small quantities under natural condition, mutation and selection processes have been employed for the improvement of strains. Recently, metabolic engineering strategies have been employed for more efficient production of these bioproducts. Metabolic engineering can be defined as purposeful modification of cellular metabolic pathways by introducing new pathways, deleting or modifying the existing pathways for the enhanced production of a desired product or modified/new product, degradation of xenobiotics, and utilization of inexpensive raw materials. Metabolic flux analysis and metabolic control analysis along with recombinant DNA techniques are three important components in designing optimized metabolic pathways, This powerful technology is being further improved by the genomics, proteomics, metabolomics and bioinformatics. Complete genome sequences are providing us with the possibility of addressing complex biological questions including metabolic control, regulation and flux. In silico analysis of microbial metabolic pathways is possible from the completed genome sequences. Transcriptome analysis by employing ONA chip allows us to examine the global pattern of gene expression at mRNA level. Two dimensional gel electrophoresis of cellular proteins can be used to examine the global proteome content, which provides us with the information on gene expression at protein level. Bioinformatics can help us to understand the results obtained with these new techniques, and further provides us with a wide range of information contained in the genome sequences. The strategies taken in our lab for the production of pharmaceutical proteins, polyhydroxyalkanoate (a family of completely biodegradable polymer), succinic acid and me chemicals by employing metabolic engineering powered by genomics, proteomics, metabolomics and bioinformatics will be presented.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2002.04a
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• pp.19-25
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• 2002

Biotechnology in the 21st century will be driven by three emerging technologies: genomics, high-throughput biology, and bioinformatics. These technologies are complementary to one another. A large number of economically important crops are currently subjected to whole genome sequencing. Functional genomics for determining the functions of the genes comprising the given plant genome is under progress by using various means including phenotyping data from transgenic mutants, gene expression profiling data from DNA microarrays, and metabolic profiling data from LC/mass analysis. The aim of plant molecular breeding is shifting from introducing agronomic traits such as herbicide and insect resistance to introducing quality traits such as healthful oils and proteins, which will lead to improved and nutritional food and feed products. Plant molecular breeding is also expected to aim to develop crops for producing human therapeutic and industrial proteins.

• Journal of Plant Biotechnology
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• v.29 no.3
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• pp.145-150
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• 2002

Biotechnology in the 21st century will be driven by three emerging technologies: genomics, high-throughput biology, and bioinformatics. These technologies are complementary to one another. A large number of economically important crops are currently subjected to whole genome sequencing. Functional genomics for determining the functions of the genes comprising the given plant genome is under progress by using various means including phenotyping data from transgenic mutants, gene expression profiling data from DNA microarrays, and metabolic profiling data from LC/mass analysis. The aim of plant molecular breeding is shifting from introducing agronomic traits such as herbicide and insect resistance to introducing quality traits such as healthful oils and proteins, which will lead to improved and nutritional food and feed products. Plant molecular breeding is also expected to aim to develop crops for producing human therapeutic and industrial proteins.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.135-137
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• 2008

The metabolic engineering of epothilones, as secondary metabolites, was investigated using Sorangium cellulosum to achieve the selective production of epothilone B, a potent anticancer agent. Thus, the propionyl-CoA synthetase gene (prpE) from Ralstonia solanacearum was heterologously expressed in S. cellulosum to increase the production of epothilone B. Propionyl-CoA synthetase converts propionate into propionyl-CoA, a potent precursor of epothilone B. The recombinant S. celluloslim containing the prpE gene exhibited a significant increase in the resolution of epothilones B/A, with an epothilone B to A ratio of 127 to 1, which was 100 times higher than that of the wild-type cells, demonstrating its potential use for the selective production of epothilone B.

• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.297-303
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• 2013

Phylogenetic analysis of the groEL1 and xynB1 gene sequences from Sorangium cellulosum strains isolated in Korea previously revealed the existence of at least 5 subgroups (A-E). In the present study, we used sequence analysis of polymerase chain reaction-amplified biosynthetic genes of strains from the 5 subgroups to indicate correlations between S. cellulosum subgroups and their secondary metabolic gene categories. We detected putative biosynthetic genes for disorazol, epothilone, ambruticin, and soraphen in group A, group C, group D, and group E strains, respectively. With the exception of KYC3204, culture extracts from group A, group B, and group C strains exhibited no noticeable antimicrobial inhibitory activities. By contrast, culture extracts from group D strains inhibited the growth of Candida albicans, whereas culture extracts from group E strains inhibited the growth of C. albicans and Staphylococcus aureus. High performance liquid chromatography analysis of the culture extracts from the strains of each subgroup revealed unique peak patterns. Our findings indicate the existence of at least 5 subgroups of S. cellulosum strains, each of which has the potential to produce a unique set of secondary metabolites.

• Journal of Plant Biotechnology
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• v.36 no.3
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• pp.294-300
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• 2009

Activation tagging that uses T-DNA vectors containing multimerized transcriptional enhancers from the cauliflower mosaic virus (CaMV) 35S gene is a powerful tool to determine gene function in plants. This approach has been successfully applied in screening various types of mutations and cloning the corresponding genes. We generated an activation tagged hairy root pool of ginseng (Panax ginseng C.A. Meyer) in an attempt to isolate genes involved in the biosynthetic pathway of ginsenoside (triterpene saponin), which is known as the major active ingredient of the root. Quantitative and qualitative variation of ginsenoside in activation tagged hairy root lines were profiled using LC/MS. Metabolic profiling data enabled selection of a specific hairy root line which accumulated ginsenoside at a higher level than other lines. The relative expression level of several genes of triterpene biosynthetic pathway in the selected hairy root line was determined by real time RT-PCR. Overall results suggest that the activation tagged ginseng hairy root system described in this study would be useful in isolating genes involved in a complex metabolic pathway from genetically intractable plant species by metabolic profiling.

• Korean Journal of Microbiology
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• v.54 no.3
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• pp.175-187
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• 2018

Myxobacteria produce diverse secondary metabolites for predation, self-defense, intercellular signaling, and other unknown functions. Many secondary metabolites isolated from myxobacteria show pharmaceutically useful bioactivity such as anticancer, antibacterial, and antifungal activities with a unique mechanism of action. Therefore, a large number of myxobacterial strains have been isolated globally and many bioactive compounds have been purified from them. However, 16S rRNA database analysis indicates that there are far more types of myxobacterial species in the wild than have ever been isolated, and genome sequence analysis suggests that each myxobacterium is capable of producing much more metabolites than already known. In this article, the current status of studies on the secondary metabolites from myxobacteria, their biosynthetic genes, biological functions, and transcriptional regulatory factors governing gene expression were reviewed.

• Journal of Life Science
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• v.24 no.10
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• pp.1055-1062
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• 2014

We investigated the expression of cucumber genes involved in lipid mobilization and metabolism during cotyledon development to compare gene activity and to study the direction of carbon (acetyl unit) transport between glyoxysomes and mitochondria. The core metabolic pathway involving 10 genes was examined in four intracellular compartments: glyoxysomes (peroxisomes), mitochondria, chloroplasts, and cytosol. Additionally, we tested the early germination response of dark-grown seedlings and the immediate light response for a further 3 days. According to the reverse transcription polymerase chain reaction (RT-PCR), 3-L-ketoacyl-CoA thiolase 2 (Thio2), isocitrate lyase (ICL), and malate synthase (MS), the genes involved in storage lipid mobilization showed a similar and consistent pattern of gene expression in seedling development. Furthermore, coordinate expression of the A BOUT DE SOUFFLE (BOU) gene with ICL and MS during seedling emergence pointed to a possible secondary route of acetyl unit (acetyl-CoA) transport between peroxisomes and mitochondria in cucumber. The expression of the BOU gene was light dependent, as shown by BOU activity in Arabidopsis, suggesting that the dark condition also results in weak membrane biogenesis. In addition, several genes were active throughout the development of the green cotyledon, even during senescence. In conclusion, this study summarizes oil-seed germination and gene expression during cucumber cotyledon development and proposes an additional route for acetyl unit transport.