• Journal of Microbiology and Biotechnology
• /
• 제16권7호
• /
• pp.1111-1119
• /
• 2006

Beauvericins and enniatins are cyclohexadepsipeptides exhibiting various biological activities on animal systems, including humans. Fusarium oxysporum FB1501 (KFCC 11363P) that produces four different cyclohexadepsipeptides was isolated from soil in Korea and the structures of the four cyclohexadepsipeptides elucidated by HPLC, MS, IR, and NMR analyses. The molecular weights for compounds 1,2,3, and 4 were determined to be 654.5, 784.5, 668.6, and 682.5, respectively, on the basis of ESI-MS measurements. The IR spectra for all the compounds exhibited absorptions for ester $(1,733-1,743\;cm^{-1})$ and amide $(1,649-1,655\;cm^{-1})$ bonds that were very similar to those for beauvericin and enniatins with ester and amide absorptions. The results of the NMR analysis $(^{1}H,\;^{13}C,\;135-DEPT,\;COSY,\;HMQC,\;and\;HMBC;\;in\;COCl_{3})$ revealed that compounds 1,3, and 4 consisted of $_{L}-N-methyl\;valine$ (N-MeVal), $_{D}-{\alpha}-hydroxyisovaleic\;acid$ (Hiv), and 2-hydroxy-3-methylpentanoic acid (Hmp) residues (compound 1: three N-MeVal residues, two Hiv residues, and one Hmp residue; compound 3: three N-MeVal residues, one Hiv, and two Hmp residues; compound 4: three N-MeVal residues and three Hmp residues). Therefore, the compounds were identified as enniatin H (compound 1), enniatin I (compound 3), and enniatin MK1688 (compound 4). Compound 2 was analyzed as beauvericin according to 1D and 2D NMR analyses. This study is the first report related to the co-production of beauvericin with other unusual enniatins, such as enniatin H, enniatin I, and enniatin MK1688, by Fusarium oxysporum.

• 한국식물병리학회지
• /
• 제12권1호
• /
• pp.72-79
• /
• 1996

보리로부터 분리한 Fusarium sp. BYA-1균주의 감자한천배지 배양체로부터 여러 식물병원곰팡이에 길항력을 나타내는 세 개의 항생물질을 분리하였다. 추출한 세 개의 항생물질은 silica gel관 크로마토그래피와 분취 HPLC, 그리고 Phytolhthora capsici 검정을 이용하여 정제하였다. 이들 분리한 항생물질들을 동정하기 위하여 융점 결정, 자외선흡광법, 질량분석 및 핵자기공명법 등의 기기분석을 실시하였다. 그 결과, 세 개의 항진균성 항생물질들은 fusarielin A, enniatin B, 그리고 enniatin B\ulcorner으로 각각 동정되었다. 분리한 세 개의 물질 중 fusarielin A가 공시된 곰팡이에 가장 강한 항균활성을 나타내었으며, 최소저해농도는 40$\mu\textrm{g}$/ml이하였다. Fusarium속 균주가 구조적으로 다른 두 종류의 항진균성 항생물질인 fusarielin A와 enniatins을 동시에 생성한다는 것은 본 논문에서 처음으로 보고하는 것이다.

• 한국식품위생안전성학회지
• /
• 제23권1호
• /
• pp.73-79
• /
• 2008

Beauvericin과 enniatin H, I, 그리고 MK1688의 생산에 미치는 온도와 수분함량의 효과를 조사하였다. 쌀을 기질로 할 경우 $25^{\circ}C$, 40% 수분함량에서 조사된 모든 독소들이 최대로 생산되었으며, $15^{\circ}C$의 온도에서 접종 2주 후 $50\;{\mu}g/g$이하의 생성량을 나타내었다. 수분함량 10%에서도 접종 후 6주차에 모든 독소들의 검출이 확인되었으며, 이는 이 독소들이 0.75정도의 낮은 수분활성도에서도 생성될 수 있음을 나타낸다. 한편, 국내에서 생산된 곡류들(65종)에 대하여 Fusarium 독소인 beauvericin의 오염을 분석하였다. 국내에서 수확된 65종의 곡류시료 중 6종의 시료에서 beauvericin오염이 확인되었다. 쌀과 현미에서는 beauvericin이 검출되지 않았으며, 2004년산 옥수수 3종, 2004년과 2005산 보리 시료 각 1종, 그리고 2005년 재배된 밀 1종에서 beauvericin이 검출되었다. 가장 높은 오염도를 보인 것은 옥수수 시료이며, 이 시료에서 $0.23\;{\mu}g/g$의 beauvericin이 검출되었다. 국내에서는 beauvericin에 대한 곡류 오염 조사가 이 연구에서 처음으로 이루어졌으며, 국내산 곡류에서도 beauvercin이 검출됨에 따라 지속적으로 오염도를 조사할 필요가 있다.

• BMB Reports
• /
• 제29권6호
• /
• pp.493-499
• /
• 1996

The biochemical properties of purified D-hydruxyisovalerate dehydrogenase from Fusarium sambucinum was elucidated. D-Hydroxyisovalerate dehydrogenase produced solely D-hydroxyisovalerate from 2-ketoisovalerate. The isoelectric point of the purified enzyme was 7.0. The enzyme was highly specific with 2-ketoisovalerate ($K_{m}=0.188$ mM, $V_{max}=8.814$ mmol/min mg) and 2-keto-3-methyl-n-valerate ($K_{m}=0.4$ mM, $V_{max}=1.851$ mmol/min mg) for the reductive reaction. This was also seen by comparing D-hydroxyisovalerate ($K_{m}=1.667$ mM, $V_{max}=0.407$ mmol/min mg) and D-hydroxy-3-methyl-n-valerate ($K_{m}=6.7$ mM, $V_{max}=0.648$ mmol/min mg) for the oxidative reaction. Thiol blocking reagents, such as iodoacetamide, N-ethylmaleimide and p-chloromecuribenzoate inhibited about 80% of enzyme activity at 0.02 mM, 50 mM and 50 mM, respectively. The enzyme activity was also inhibited by the addition of 0.1 mM of various metal ions, such as $Fe^{2+}$ (67%), $Cu^{2+}$ (88%), $Zn^{2+}$ t (76%) and $Mg^{2+}$ (9%). The enzyme was stable over three months in 50 mM potassium phosphate buffer (pH 5~7) at $-80^{\circ}C$. However the purified enzyme lost 30% of its activity in the same buffer after 24 h at $4^{\circ}C$. The studies about thermal inactivation of D-hydroxyisovalerate dehydrogenase exhibit 209.2 kJ/M of activation enthalpy and 0.35 kJ/mol K of activation entropy.

• 한국식품위생안전성학회지
• /
• 제37권5호
• /
• pp.347-355
• /
• 2022

본 연구에서는 국내산 생강(n = 43)과 생강가루(n = 31)를 대상으로 beauvericin (BEA)과 enniatins (ENNs)의 오염 실태를 조사하였다. 생강 시료 중 62.79%가 BEA에 오염되었으며, 최대 오염농도는 640.07 ㎍/kg으로 오염률과 오염농도가 조사 대상 독소 중 가장 높았다. 생강에서 ENNs의 오염률은 최대 11.63% (ENB, ENB1)이었으며, 최대 오염농도는 91.02 ㎍/kg (ENA)였다. 생강가루에서는 ENB의 오염률이 70.97%로 가장 높았으나, 오염농도는 BEA이 최대 1, 344.18 ㎍/kg으로 조사 대상 독소 중 가장 높았다. 생강가루의 ENA, ENA1, ENB, ENB1의 오염률은 29.03%, 22.58%, 70.97%, 35.48%였으며, 최대 오염농도는 220.45 ㎍/kg, 156.61 ㎍/kg, 413.99 ㎍/kg, 70.29 ㎍/kg로 ENB의 오염률과 오염농도가 높았다. BEA과 ENNs은 생강보다 생강가루에서 오염농도가 높았다. 생강과 생강가루에서 BEA과 ENNs의 중복 오염률은 각각 16.28%와 64.52%로 생강에 비해 생강가루에서 독소의 중복오염률이 높았다. 본 연구는 한국산 생강과 생강가루의 BEA과 ENNs의 발생 및 이들의 중복 오염을 처음으로 보고하는 것이다.

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