• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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• pp.236-241
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• 2004

In Escherichia coli, L-lysine biosynthetic pathway is composed of nine enzymatic reactions. It has been well established that most of the lysine biosynthetic genes are regulated by the lysine availability, even though they are all scattered around the chromosome without forming any multigenic operon structure. However, no transcriptional regulatory mechanism has been identified except for the activation of lysA gene by the LysR. In this study, changes in transcriptome profiles of wild type cells and lysR deletion mutant cells grown in the absence or presence of lysine were investigated by using DNA microarray technique. Microarray data analysis revealed three groups of genes whose expression varies depending on the availability of lysine or LysR or both. To further examine the regulatory patterns of lysine biosynthetic genes, lacZ operon fusions were constructed and their expression was measured under various conditions. Obtained results strongly suggest that there is an additional regulatory mechanism which senses the lysine availability and coordinates gene expression.

• Journal of Microbiology and Biotechnology
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• v.29 no.12
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• pp.1931-1937
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• 2019

The heterologous expression of the Streptomyces natural product (NP) biosynthetic gene cluster (BGC) has become an attractive strategy for the activation, titer improvement, and refactoring of valuable and cryptic NP BGCs. Previously, a Streptomyces artificial chromosomal vector system, pSBAC, was applied successfully to the precise cloning of large-sized polyketide BGCs, including immunosuppressant tautomycetin and antibiotic pikromycin, which led to stable and comparable production in several heterologous hosts. To further validate the pSBAC system as a generally applicable heterologous expression system, the daptomycin BGC of S. roseosporus was cloned and expressed heterologously in a model Streptomyces cell factory. A 65-kb daptomycin BGC, which belongs to a non-ribosomal polypeptide synthetase (NRPS) family, was cloned precisely into the pSBAC which resulted in 28.9 mg/l of daptomycin and its derivatives in S. coelicolor M511(a daptomycin non-producing heterologous host). These results suggest that a pSBAC-driven heterologous expression strategy is an ideal approach for producing low and inconsistent Streptomyces NRPS-family NPs, such as daptomycin, which are produced low and inconsistent in native host.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.66 no.4
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• pp.365-374
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• 2021

Soybean isoflavones are essential secondary metabolites synthesized through the phenylpropanoid pathway, and they play vital roles in human health. Isoflavone content is a complex quantitative trait controlled by multiple genes, and the genetic mechanisms underlying isoflavone biosynthesis remain largely unknown. Therefore, the present study analyzed the content of isoflavone and expression of six key genes involved in its biosynthesis (i.e., CHS6, HID, IF7GT, IF7MaT, GmIMaT1, and GmIMaT3) during soybean seed germination. Isoflavone content was quantified using high-performance liquid chromatography, and isoflavone biosynthetic gene expression was analyzed using quantitative real-time PCR. Two cultivars, namely 'Daepung2ho' and 'Pungsannamulkong', which are high- and low-isoflavone cultivars, respectively, were used. Isoflavone accumulation gradually increased with the progression of the germination period. As such, malonyl glucosides accounted for over 80% of the total content, whereas acetyl glucosides were present at trace amounts. Transcriptional analysis of isoflavone biosynthetic genes demonstrated expression patterns parallel to isoflavone content; however, there was no clear correlation between isoflavone content and gene expression. Moreover, most isoflavone biosynthetic genes showed different expression patterns depending on the individual gene or genotypes. Among the tested genes, HID showed consistently higher expression, except at 3 days after germination, and its expression was upregulated in 'Daepung2ho' but downregulated in 'Pungsannamulkong'. In addition, all tested genes exhibited different expression patterns between cotyledons and hypocotyls and responded differently to the germination period. These findings suggest that the expression levels of isoflavone biosynthetic genes are not consistent with the germination period and appear to be genotype-dependent.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.828-836
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• 2008

The biocontrol rhizobacterium Pseudomonas sp. M18 can produce two kinds of antibiotics, namely pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA), and is antagonistic against a number of soilborne phytopathogens. In this study, a luxR-type quorum-sensing regulatory gene, vqsR, was identified and characterized immediately downstream of the Plt gene cluster in strain MI8. A vqsR-inactivated mutant led to a significant decrease in the production of Plt and its biosynthetic gene expression. However, this was restored when introducing the vqsR gene by cloning into the plasmid pME6032 in trans. The vqsR mutation did not exert any obvious influence on the production of PCA and its biosynthetic gene expression and the production of N-acylhomoserine lactones (C4 and C8-HSLs) and their biosynthetic gene rhlI expression. Accordingly, these results introduce VqsR as a regulator of Plt production in Pseudomonas spp., and suggest that the regulatory mechanism of vqsR in strain M18 is distinct from that in P. aeruginosa. In addition, it was demonstrated that vqsR mutation did not have any obvious impact on the expression of Plt-specific ABC transporters and other secondary metabolic global regulators, including GacA, RpoS, and RsmA.

• The Plant Pathology Journal
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• v.39 no.5
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• pp.417-429
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• 2023

Ralstonia solanacearum species complex (RSSC) is a soil borne plant pathogen causing bacterial wilt on various important crops, including Solanaceae plants. The bacterial pathogens within the RSSC produce exopolysaccharide (EPS), a highly complicated nitrogencontaining heteropolymeric polysaccharide, as a major virulence factor. However, the biosynthetic pathway of the EPS in the RSSC has not been fully characterized. To identify genes in EPS production beyond the EPS biosynthetic gene operon, we selected the EPS-defective mutants of R. pseudosolanacearum strain SL341 from Tn5-inserted mutant pool. Among several EPSdefective mutants, we identified a mutant, SL341P4, with a Tn5-insertion in a gene encoding a putative NDP-sugar epimerase, a putative membrane protein with sugar-modifying moiety, in a reverse orientation to EPS biosynthesis gene cluster. This protein showed similar to other NDP-sugar epimerases involved in EPS biosynthesis in many phytopathogens. Mutation of the NDP-sugar epimerase gene reduced EPS production and biofilm formation in R. pseudosolanacearum. Additionally, the SL341P4 mutant exhibited reduced disease severity and incidence of bacterial wilt in tomato plants compared to the wild-type SL341 without alteration of bacterial multiplication. These results indicate that the NDP-sugar epimerase gene is required for EPS production and bacterial virulence in R. pseudosolanacearum.

• Journal of Microbiology and Biotechnology
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• v.19 no.5
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• pp.439-446
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• 2009

The biosynthesis study of antibiotics saframycin (SFM) in Streptomyces lavendulae and safracin (SAC) in Pseudomonas fluorescens demonstrated that 3-hydroxy-S-methyl-O-methyltyrosine (3hSmOmTyr), a nonproteinogenic amino acid, is the precursor of the tetrahydroisoquinoline molecular core. In the biosynthetic gene cluster of SAC/SFM, sacD/sfmD encodes a protein with high homology to each other but no sequence similarity to other known enzymes; sacF/sfmM2 and sacG/sfmM3 encode methyltransferases for C-methylation and O-methylation; and sacE/sfinF encodes a small protein with significant sequence similarity to the MbtH-like proteins, which are frequently found in the biosynthetic pathways of non ribosomal peptide antibiotics and siderophores. To address their function, the biosynthetic cassette of 3h5mOmTyr was heterologously expressed in S. coelicolor and P. putida, and an in-frame deletion and complementation in trans were carried out. The results revealed that (i) SfmD catalyzes the hydroxylation of aromatic rings; (ii) sacD/sacF/sacG in the SAC gene cluster and sfmD/sfmM2/sfmM3 in the SFM cluster are sufficient for the biosynthesis of 3h5mOmTyr; and (iii) the mbtH-like gene is not required for the biosynthesis of the 3h5mOmTyr precursor.

• Molecules and Cells
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• v.20 no.1
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• pp.90-96
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• 2005

A cluster of genes for ribostamycin (Rbm) biosynthesis was isolated from Streptomyces ribosidificus ATCC 21294. Sequencing of 31.892 kb of the genomic DNA of S. ribosidificus revealed 26 open reading frames (ORFs) encoding putative Rbm biosynthetic genes as well as resistance and other genes. One of ten putative Rbm biosynthetic genes, rbmA, was expressed in S. lividans TK24, and shown to encode 2-deoxy-scyllo-inosose (DOI) synthase. Acetylation of various aminoglycoside-aminocyclitol (AmAcs) by RbmI confirmed it to be an aminoglycoside 3-N-acetyltransferase. Comparison of the genetic control of ribostamycin and butirosin biosynthesis pointed to a common biosynthetic route for these compounds, despite the considerable differences between them in genetic organization.

• Mycobiology
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• v.44 no.2
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• pp.105-111
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• 2016

Paclitaxel (taxol) has long been used as a potent anticancer agent for the treatment of many cancers. Ever since the fungal species Taxomyces andreanae was first shown to produce taxol in 1993, many endophytic fungal species have been recognized as taxol accumulators. In this study, we analyzed the taxol-producing capacity of different Colletotrichum spp. to determine the distribution of a taxol biosynthetic gene within this genus. Distribution of the taxadiene synthase (TS) gene, which cyclizes geranylgeranyl diphosphate to produce taxadiene, was analyzed in 12 Colletotrichum spp., of which 8 were found to contain the unique skeletal core structure of paclitaxel. However, distribution of the gene was not limited to closely related species. The production of taxol by Colletotrichum dematium, which causes pepper anthracnose, depended on the method in which the fungus was stored, with the highest production being in samples stored under mineral oil. Based on its distribution among Colletotrichum spp., the TS gene was either integrated into or deleted from the bacterial genome in a species-specific manner. In addition to their taxol-producing capacity, the simple genome structure and easy gene manipulation of these endophytic fungal species make them valuable resources for identifying genes in the taxol biosynthetic pathway.

• Korean Journal of Plant Tissue Culture
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• v.25 no.5
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• pp.323-327
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• 1998

The flavonoid biosynthetic pathway has been studied as a genetic model system, particularly in Petunia hybrida. In order to study the flavonoid biosynthetic pathway, we constructed a fusion gene system between Cauliflower Mosaic Virus (CaMV) 35S promoter and eggplant flavonoid 3', 5'-hydroxylase in pBI 121 plasmid. An optimal condition for plant regeneration was observed when internode explants were cultured on MS medium supplemented with IAA 0.2 mg/L plus BA 3 mg/L. For plant transformation internode explants of Petunia hybrida were precultured on BM medium supplemented with IAA 0.2 mg/L plus BA 3 mg/L. Putative transgenic plants were selected on medium containing kanamycin 50 mg/L plus cefotaxim 300 mg/L. Putative selected transformants were confirmed by amplification of selectable marker gene (nptII) by polymerase chain reaction (PCR) and Southern hybridization of flavonoid 3',5'-hydroxylase gene.

• Microbiology and Biotechnology Letters
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• v.49 no.3
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• pp.458-465
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• 2021

Tubulysins are a group of bioactive secondary metabolites from myxobacteria exhibiting strong anticancer activity against various cancer cell lines. In this study, we describe the identification of putative tubulysin biosynthetic gene clusters (tubA~tubF) in the genome sequences of two tubulysin-producing myxobacterial strains, Archangium gephyra MEHO_002 and MEHO_004. The inactivation of the putative tubulysin biosynthetic genes resulted in a tubulysin-production defect. The DNA sequences of the A. gephyra MEHO_002 and MEHO_004 tubulysin biosynthetic genes were 97% identical, and the amino acid sequences of the encoded proteins shared a similarity of 97-100%. The nucleotide sequences of the tubulysin biosynthetic gene clusters in MEHO_002 and MEHO_004 were 86% identical to that in Cystobacter sp. SBCb004 known as a tubulysin-producing myxobacterium, and the organization of the clusters was identical except for the lack of a tubZ gene in the clusters in MEHO_002 and MEHO_004. The amino acid sequences of the proteins encoded by each gene were 88-97% similar to those encoded by SBCb004, and the domain compositions of the proteins were also identical.