• Journal of Microbiology and Biotechnology
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• v.16 no.7
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• pp.1111-1119
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• 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.

• Korean Journal Plant Pathology
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• v.12 no.1
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• pp.72-79
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• 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.

• Journal of Food Hygiene and Safety
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• v.23 no.1
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• pp.73-79
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• 2008

The productions of beauvericin and enniatins H, I, and MK1688 by Fusarium oxysporum KFCC 11363P were investigated on rice substrate at four temperatures (15, 20, 25, and $30^{\circ}C$) and three moisture contents (10, 20, and 40%). The largest amount of beauvericin ($718.0\;{\mu}g/g$) was produced at $25^{\circ}C$, and maximum levels of enniatin H ($781.9\;{\mu}g/g$), I ($725.8\;{\mu}g/g$), and MK1688 ($425.8\;{\mu}g/g$) were measured by high pressure liquid chromatography (HPLC) at the same temperature. The optimal moisture content for the production of beauvericin and enniatins H, I, and MK1688 was 40%, and the trace amounts of these toxins were observed at 10% moisture content. Sixty five grain samples (n=65) were tested for the monitoring of beauvericin. This mycotoxin was detected in six grain samples including three maize, two barley, and one wheat samples. The highest contamination level of beauvericin was observed in maize sample ($0.23\;{\mu}g/g$).

• BMB Reports
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• v.29 no.6
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• pp.493-499
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• 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.

• Journal of Food Hygiene and Safety
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• v.37 no.5
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• pp.347-355
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• 2022

Levels of beauvericin (BEA) and enniatins (ENNs: ENA, ENA1, ENB, and ENB1) were examined in fresh ginger (n = 43) and ginger powder (n = 31) samples from Korea. In the ginger samples, incidence of BEA contamination was highest, at 62.79%, with a maximum detected BEA level of 640.07 ㎍/kg. ENNs in were found in up to 11.63% (ENB, ENB1) of ginger samples, with a maximum detected level of 91.02 ㎍/kg (ENA). In the ginger powders, ENB contamination displayed the highest rate of incidence (70.97%), but the highest level of BEA (1,344.18 ㎍/kg) exceeded that of ENB (413.99 ㎍/kg). The incidences of ENA, ENA1, ENB, and ENB1 presence in ginger powders were 29.03%, 22.58%, 70.97%, and 35.48%, respectively, and their highest detected levels were 220.45, 156.61, 413.99, and 70.29 ㎍/kg, respectively. The incidence of BEA and ENN contamination was higher in ginger powder than in ginger. Respective co-occurrence rates of BEA and ENNs in ginger and ginger powder samples were 16.28% and 64.52%, indicating that the BEA and ENN co-contamination rate was highest in ginger powder as well. This is the first report on the presence and co-occurrence of BEA and ENNs in Korean ginger and ginger powder.

• 한국균학회소식:학술대회논문집
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• 2014.05a
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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.