• Journal of Applied Biological Chemistry
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• v.65 no.3
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• pp.159-166
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• 2022

Epicoccum nigrum produces epipyrone A (orevactaene), a yellow polyketide pigment. Its biosynthetic gene cluster was previously characterized in E. nigrum KACC 40642. The YES liquid culture of this strain revealed high-level production of epicoccamide (EPC), with an identity that was determined using liquid chromatography-mass spectrometry analysis and molecular mass search using the SuperNatural database V2 webserver. The production of EPC was further confirmed by compound isolation and nuclear magnetic resonance spectroscopy. EPC is a highly reduced polyketide with tetramic acid and mannosyl moieties. The EPC structure guided us to localize the hypothetical EPC biosynthetic gene cluster (BGC) in E. nigrum ICMP 19927 genome sequence. The BGC contains genes encoding highly reducing (HR)-fungal polyketide synthase (fPKS)-nonribosomal peptide synthetase (NRPS), glycosyltransferase (GT), enoylreductase, cytochrome P450, and N-methyltrasnferase. Targeted inactivation of the HR-fPKS-NRPS and GT genes abolished EPC production, supporting the successful localization of EPC BGC. This study provides a platform to explore the hidden biological activities of EPC, a bolaamphiphilic compound.

• Journal of Microbiology and Biotechnology
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• v.26 no.9
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• pp.1636-1642
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• 2016

Phenalamide is a bioactive secondary metabolite produced by Myxococcus stipitatus. We identified a 56 kb phenalamide biosynthetic gene cluster from M. stipitatus DSM 14675 by genomic sequence analysis and mutational analysis. The cluster is comprised of 12 genes (MYSTI_04318- MYSTI_04329) encoding three pyruvate dehydrogenase subunits, eight polyketide synthase modules, a non-ribosomal peptide synthase module, a hypothetical protein, and a putative flavin adenine dinucleotide-binding protein. Disruption of the MYSTI_04324 or MYSTI_04325 genes by plasmid insertion resulted in a defect in phenalamide production. The organization of the phenalamide biosynthetic modules encoded by the fifth to tenth genes (MYSTI_04320-MYSTI_04325) was very similar to that of the myxalamid biosynthetic gene cluster from Stigmatella aurantiaca Sg a15, as expected from similar backbone structures of the two substances. However, the loading module and the first extension module of the phenalamide synthase encoded by the first to fourth genes (MYSTI_04326-MYSTI_04329) were found only in the phenalamide biosynthetic gene cluster from M. stipitatus DSM 14675.

• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1101-1108
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• 2008

Geldanamycin and its analogs are important anticancer agents that inhibit the newly targeted heat-shock protein (Hsp) 90, which is a chaperone protein in eukaryotic cells. To resolve which geldanamycin biosynthetic genes are responsible for particular post-polyketide synthase (PKS) processing steps and in which order the reactions occur, we individually inactivated candidate genes in Streptomyces hygroscopicus subsp. duamyceticus JCM4427 and isolated and elucidated the structures of intermediates from each mutant. The results indicated that gel7 governs at least one of the benzoquinone ring oxidation steps. The gel16 was found to be involved in double-bond formation between C-4 and C-5 of 4,5-dihydrogeldanamycin, which confirmed our previous findings that this double bond is reduced during the post-PKS modification of the polyketide assembly. In addition, pro-geldanamycin, which does not possess a double bond at C-4/5, was purified from the gel7 and gel8 double-gene-inactivated mutant.

• KSBB Journal
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• v.24 no.2
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• pp.163-169
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• 2009

Lovastatin (also known as Mevinolin, Mevacor, and Monacolin K), an inhibitor of the HMG-CoA reductase produced by Aspergillus terreus and other fungi, is used to reduce serum cholesterol levels in human beings. It is derived biosynthetically from two polyketides. One of these is a nonaketide that undergoes cyclization at a hexahydronaphthalene ring system, and the other is a simple diketide, 2-methylbutyrate. Two primer pairs were designed based on the amino acid sequences of lovastatin polyketide synthase and lovastatin diketide synthase for the PCR screening of lovastatin-producing strains. Among the seven selected strains, SJ-2 evidenced the highest level of lovastatin production in both liquid and solid cultures. Soybeans with SJ-2 were treated via 1 hour of heat shock at $30^{\circ}C$ for the mass production of lovastatin. The heat-treated soybeans were inoculated on rice bran and the koji extract was obtained after 15 days of incubation. It yielded the highest level of lovastatin production among the strains, and also evidenced 75% inhibition activity against HMG-CoA reductase. We developed an efficient PCR screening method for lovastatin-producing strains, using lovastatin biosynthesis genes.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.420-422
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• 2005

TMC (Tautomycetin) is a liner polyketide immunosuppressive antifungal compound produced by Streptomyces spp. Inhibition of T cell proliferation with TMC was observed highly efficient at 100-fold lower than those needed to achieve maximal inhibition with cyclosporin A. To elucidate the biosynthetic pathway of TMC, a genomic DNA library was constructed using a E. coil-Streptomyces shuttle cosmid vector, pOJ446. The DNA libraries were screened by colony blot hybridization using several polyketide ${\beta}-ketosynthase$ (KS) probes amplified from TMC-producing Streptomyces genomic DNA using polymerase chain reaction (PCR), of which the degenerate primers were designed based on the highly conserved sequences present in KS domains of various type I polyketide synthase genes in Streptomyces species. This library construction and screening approach led to the isolation of several positive cosmid clones representing type I polyketide biosynthetic gene clusters. In addition, a Streptomyces regulatory gene called afsR2 (a global regulatory gene stimulating antibiotic production in both S. coelicolor and S. lividans) was successfully integrated into the TMC-producing Streptomyces chromosome via E. coil-Streptomyces heterologous conjugation mehtod. The more detailed results of production improvement and genetic characterization of TMC-producing Streptomyces spp. will be discussed.

• 한국균학회소식:학술대회논문집
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• 2016.05a
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• pp.27-27
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• 2016

The ascomycete fungus Fusarium graminearum is the most common pathogen of Fusarium head blight (FHB), a devastating disease for major cereal crops worldwide. FHB causes significant crop losses by reducing grain yield and quality as well as contaminating cereals with trichothecenes and zearalenone (ZEA) that pose a serious threat to animal health and food safety. ZEA is a causative agent of hyperestrogenic syndrome in mammals and can result in reproductive disorders in farm animals. In F. graminearum, the ZEA biosynthetic cluster is composed of four genes, PKS4, PKS13, ZEB1, and ZEB2, which encode a reducing polyketide synthase, a nonreducing polyketide synthase, an isoamyl alcohol oxidase, and a transcription factor, respectively. Although it is known that ZEB2 primarily acts as a regulator of ZEA biosynthetic cluster genes, the mechanism underlying this regulation remains undetermined. In this study, two isoforms (ZEB2L and ZEB2S) from the ZEB2 gene in F. graminearum were characterized. It was revealed that ZEB2L contains a basic leucine zipper (bZIP) DNA-binding domain at the N-terminus, whereas ZEB2S is an N-terminally truncated form of ZEB2L that lacks the bZIP domain. Interestingly, ZEA triggered the induction of both ZEB2L and ZEB2S transcription. In ZEA producing condition, the expression of ZEB2S transcripts via alternative promoter usage was directly or indirectly initiated by ZEA. Physical interaction between ZEB2L and ZEB2L as well as between ZEB2L and ZEB2S was observed in the nucleus. The ZEB2S-ZEB2S interaction was detected in both the cytosol and the nucleus. ZEB2L-ZEB2L oligomers activated ZEA biosynthetic cluster genes, including ZEB2L. ZEB2S inhibited ZEB2L transcription by forming ZEB2L-ZEB2S heterodimers, which reduced the DNA-binding activity of ZEB2L. This study provides insight into the autoregulation of ZEB2 expression by alternative promoter usage and a feedback loop during ZEA production.

• Journal of Microbiology and Biotechnology
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• v.29 no.7
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• pp.1144-1154
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• 2019

There have been several studies regarding lichen-associated bacteria obtained from diverse environments. Our screening process identified 49 bacterial species in two lichens from the Himalayas: 17 species of Actinobacteria, 19 species of Firmicutes, and 13 species of Proteobacteria. We discovered five types of strong antimicrobial agent-producing bacteria. Although some strains exhibited weak antimicrobial activity, NP088, NP131, NP132, NP134, and NP160 exhibited strong antimicrobial activity against all multidrug-resistant strains. Polyketide synthase (PKS) fingerprinting revealed results for 69 of 148 strains; these had similar genes, such as fatty acid-related PKS, adenylation domain genes, PfaA, and PksD. Although the association between antimicrobial activity and the PKS fingerprinting results is poorly resolved, NP160 had six types of PKS fingerprinting genes, as well as strong antimicrobial activity. Therefore, we sequenced the draft genome of strain NP160, and predicted its secondary metabolism using antiSMASH version 4.2. NP160 had 46 clusters and was predicted to produce similar secondary metabolites with similarities of 5-100%. Although NP160 had 100% similarity with the alkylresorcinol biosynthetic gene cluster, our results showed low similarity with existing members of this biosynthetic gene cluster, and most have not yet been revealed. In conclusion, we expect that lichen-associated bacteria from the Himalayas can produce new secondary metabolites, and we found several secondary metabolite-related biosynthetic gene clusters to support this hypothesis.

• Journal of Microbiology and Biotechnology
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• v.17 no.3
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• pp.534-538
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• 2007

Avermectin and its analogs are major commercial antiparasitic agents in the fields of animal health, agriculture, and human infections. To increase our understanding about the genetic mechanism underlying avermectin overproduction, comparative transcriptomes were analyzed between the low producer S. avermitilis ATCC31267 and the high producer S. avermitilis ATCC31780 via a S. avermitilis whole genome chip. The comparative transcriptome analysis revealed that fifty S. avermitilis genes were expressed at least two-fold higher in S. avermitilis ATCC31780. In particular, all the avermectin biosynthetic genes, including polyketide synthase (PKS) genes and an avermectin pathway-specific regulatory gene, were less expressed in the low producer S. avermitilis ATCC31267. The present results imply that avermectin overproduction in S. avermitilis ATCC31780 could be attributed to the previously unidentified fifty genes reported here and increased transcription levels of avermectin PKS genes.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1484-1490
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• 2010

In the Streptomyces hygroscopicus JCM4427 geldanamycin biosynthetic gene cluster, five putative regulatory genes were identified by protein homology searching. Among those genes, gel14, gel17, and gel19 are located downstream of polyketide synthase genes. Gel14 and Gel17 are members of the LAL family of transcriptional regulators, including an ATP/GTP-binding domain at the N-terminus and a DNA-binding helix-turn-helix domain at the C-terminus. Gel19 is a member of the TetR family of transcriptional regulators, which generally act to repress transcription. To verify the biological significance of the putative regulators in geldanamycin production, they were individually characterized by gene disruption, genetic complementation, and transcriptional analyses. All three genes were confirmed as positive regulators of geldanamycin production. Specifically, Gel17 and Gel19 are required for gel14 as well as gelA gene expression.

• Mycobiology
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• v.51 no.1
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• pp.36-48
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• 2023

Xanthoria elegans is a lichen symbiosis, that inhabits extreme environments and can absorb UV-B. We reported the de novo sequencing and assembly of X. elegans genome. The whole genome was approximately 44.63 Mb, with a GC content of 40.69%. Genome assembly generated 207 scaffolds with an N50 length of 563,100 bp, N90 length of 122,672 bp. The genome comprised 9,581 genes, some encoded enzymes involved in the secondary metabolism such as terpene, polyketides. To further understand the UV-B absorbing and adaptability to extreme environments mechanisms of X. elegans, we searched the secondary metabolites genes and gene-cluster from the genome using genome-mining and bioinformatics analysis. The results revealed that 7 NR-PKSs, 12 HR-PKSs and 2 hybrid PKS-PKSs from X. elegans were isolated, they belong to Type I PKS (T1PKS) according to the domain architecture; phylogenetic analysis and BGCs comparison linked the putative products to two NR-PKSs and three HR-PKSs, the putative products of two NR-PKSs were emodin xanthrone (most likely parietin) and mycophelonic acid, the putative products of three HR-PKSs were soppilines, (+)-asperlin and macrolactone brefeldin A, respectively. 5 PKSs from X. elegans build a correlation between the SMs carbon skeleton and PKS genes based on the domain architecture, phylogenetic and BGC comparison. Although the function of 16 PKSs remains unclear, the findings emphasize that the genes from X. elegans represent an unexploited source of novel polyketide and utilization of lichen gene resources.