• 한국산학기술학회논문지
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• 제14권8호
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• pp.4086-4092
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• 2013

Mycosporine-like 아미노산(MAAs)은 UV 흡수물질이며, 다양한 해양생물들은 MAAs의 합성과 축적을 통하여 환경 자외선의 직 간접적인 영향을 감소시키는 기능을 진화시켜 왔다. 이 연구에서 우리는 radiofrequency(RF) 발생장치를 제작하였고, 이를 미세조류 배양에 적용하였다. $0.35{\pm}0.05$ mHz의 RF를 Chlamydomonas sp. 배양기에 공급하였고, 정해진 시간(0, 0.5, 1 및 2 시간)에 시료를 채취하였다. MAAs 생합성 관련 유전자인 dehydroquinate synthase homolog (DHQS-like)와 nonribosomal peptide synthetase homolog (NRPS-like) 유전자를 Chlamydomonas sp.로부터 클로닝하였고, RF 노출에 대한 유전자 발현을 qRT-PCR을 이용하여 분석하였다. 연구결과 RF에 노출된 Chlamydomonas sp.의 DHQS-like와 NRPS-like 유전자의 발현은 노출 1시간에 각각 1.46 배 및 1.19 배 증가하였다. 이러한 결과는 DHQS-like와 NRPS-like 유전자가 Chlaydomonas sp.의 MAAs 생합성을 진단할 수 있는 좋은 바이오마커 후보가 될 수 있음을 의미한다.

• Journal of Microbiology and Biotechnology
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• 제27권3호
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• pp.439-449
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• 2017

Beauveria bassiana infects a number of pest species and is known to produce insecticidal substances, such as the nonribosomal peptides (NRPs) beauvericin and bassianolide. However, most NRPs and their biological roles in B. bassiana remain undiscovered. To identify NRPs that potentially contribute to pathogenesis, the 21 predicted NRP synthetases (NRPSs) or NRPS-like proteins of B. bassiana ARSEF 2860 were primarily ranked into three functional groups: basic metabolism (7 NRPSs), pathogenicity (12 NRPSs), and unknown function (2 NRPSs). Based on the transcript levels during in vivo growth on diamondback moth (Plutella xylostella (Linnaeus)), half of the Group II NRPSs were likely to be involved in infection. Given that the metabolites biosynthesized by these NRPSs remain to be determined, our result underlines the importance of the NRPSome in fungal pathogenesis, and will serve as a guide for future genomic mining projects to discover functionally essential and structurally diverse NRPs in fungal genomes.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2014년도 춘계학술대회 및 임시총회
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• pp.27-28
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• 2014

Beauveria bassiana (Cordycipitaceae, Hypocreales, Ascomycota) is an anamorphic fungus having a potential to be used as a biological control agent because it parasitizes a wide range of arthropod hosts including termites, aphids, beetles and many other insects. A number of bioactive secondary metabolites (SMs) have been isolated from B. bassiana and functionally verified. Among them, beauvericin and bassianolide are cyclic depsipeptides with antibiotic and insecticidal effects belonging to the enniatin family. Non-ribosomal peptide synthetases (NRPSs) play a crucial role in the synthesis of these secondary metabolites. NRPSs are modularly organized multienzyme complexes in which each module is responsible for the elongation of proteinogenic and non-protein amino acids, as well as carboxyl and hydroxyacids. A minimum of three domains are necessary for one NRPS elongation module: an adenylation (A) domain for substrate recognition and activation; a tholation (T) domain that tethers the growing peptide chain and the incoming aminoacyl unit; and a condensation (C) domain to catalyze peptide bond formation. Some of the optional domains include epimerization (E), heterocyclization (Cy) and oxidation (Ox) domains, which may modify the enzyme-bound precursors or intermediates. In the present study, we analyzed genomes of B. bassiana and its allied species in Hypocreales to verify the distribution of NRPS-encoding genes involving biosynthesis of beauvericin and bassianolide, and to unveil the evolutionary processes of the gene clusters. Initially, we retrieved completely or partially assembled genomic sequences of fungal species belonging to Hypocreales from public databases. SM biosynthesizing genes were predicted from the selected genomes using antiSMASH program. Adenylation (A) domains were extracted from the predicted NRPS, NRPS-like and NRPS-PKS hybrid genes, and used them to construct a phylogenetic tree. Based on the preliminary results of SM biosynthetic gene prediction in B. bassiana, we analyzed the conserved gene orders of beauvericin and bassianolide biosynthetic gene clusters among the hypocrealean fungi. Reciprocal best blast hit (RBH) approach was performed to identify the regions orthologous to the biosynthetic gene cluster in the selected fungal genomes. A clear recombination pattern was recognized in the inferred A-domain tree in which A-domains in the 1st and 2nd modules of beauvericin and bassianolide synthetases were grouped in CYCLO and EAS clades, respectively, suggesting that two modules of each synthetase have evolved independently. In addition, inferred topologies were congruent with the species phylogeny of Cordycipitaceae, indicating that the gene fusion event have occurred before the species divergence. Beauvericin and bassianolide synthetases turned out to possess identical domain organization as C-A-T-C-A-NM-T-T-C. We also predicted precursors of beauvericin and bassianolide synthetases based on the extracted signature residues in A-domain core motifs. The result showed that the A-domains in the 1st module of both synthetases select D-2-hydroxyisovalerate (D-Hiv), while A-domains in the 2nd modules specifically activate L-phenylalanine (Phe) in beauvericin synthetase and leucine (Leu) in bassianolide synthetase. antiSMASH ver. 2.0 predicted 15 genes in the beauvericin biosynthetic gene cluster of the B. bassiana genome dispersed across a total length of approximately 50kb. The beauvericin biosynthetic gene cluster contains beauvericin synthetase as well as kivr gene encoding NADPH-dependent ketoisovalerate reductase which is necessary to convert 2-ketoisovalarate to D-Hiv and a gene encoding a putative Gal4-like transcriptional regulator. Our syntenic comparison showed that species in Cordycipitaceae have almost conserved beauvericin biosynthetic gene cluster although the gene order and direction were sometimes variable. It is intriguing that there is no region orthologous to beauvericin synthetase gene in Cordyceps militaris genome. It is likely that beauvericin synthetase was present in common ancestor of Cordycipitaceae but selective gene loss has occurred in several species including C. militaris. Putative bassianolide biosynthetic gene cluster consisted of 16 genes including bassianolide synthetase, cytochrome P450 monooxygenase, and putative Gal4-like transcriptional regulator genes. Our synteny analysis found that only B. bassiana possessed a bassianolide synthetase gene among the studied fungi. This result is consistent with the groupings in A-domain tree in which bassianolide synthetase gene found in B. bassiana was not grouped with NRPS genes predicted in other species. We hypothesized that bassianolide biosynthesizing cluster genes in B. bassiana are possibly acquired by horizontal gene transfer (HGT) from distantly related fungi. The present study showed that B. bassiana is the only species capable of producing both beauvericin and bassianolide. This property led to B. bassiana infect multiple hosts and to be a potential biological control agent against agricultural pests.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.251-261
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• 2017

Initially, we screened 18 Aspergillus sojae-like strains from Aspergillus spp. isolated from meju (Korean traditional fermented soybean brick) according to their morphological characteristics. Because members of Aspergillus section Flavi are often incorrectly identified because of their phylogenetic similarity, we re-identified these strains at the morphological and molecular genetic levels. Fourteen strains were finally identified as A. sojae. The isolates produced protease and ${\alpha}-amylase$ with ranges of 2.66-10.64 and 21.53-106.73 unit/g-initial dry substrate (U/g-IDS), respectively, which were equivalent to those of the koji (starter mold) strains employed to produce Japanese soy sauce. Among the isolates and Japanese koji strains, strains SMF 127 and SMF 131 had the highest leucine aminopeptidase (LAP) activities at 6.00 and 6.06 U/g-IDS, respectively. LAP plays an important role in flavor development because of the production of low-molecular-weight peptides that affect the taste and decrease bitterness. SMF 127 and SMF 131 appeared to be non-aflatoxigenic because of a termination point mutation in aflR and the lack of the polyketide synthase gene found in other A. sojae strains. In addition, SMF 127 and SMF 131 were not cyclopiazonic acid (CPA) producers because of the deletion of maoA, dmaT, and pks/nrps, which are involved in CPA biosynthesis. Therefore, A. sojae strains such as SMF 127 and SMF 131, which have high protease and LAP activities and are free of safety issues, can be considered good starters for soybean fermentations, such as in the production of the Korean fermented soybean products meju, doenjang, and ganjang.