Combinatorial Mutasynthesis of Scrambled Beauvericins,Cyclooligomer Depsipeptide Cell Migration Inhibitors from Beauveria bassiana

Fungal cyclooligomer depsipeptides such as beauvericin, bassianolide, and enniatins display antibiotic, antifungal, insecticidal, broad‐spectrum cancer cell antiproliferative, and cell migration inhibitory activities. We have identified a gene encoding a novel enzyme, ketoisovalerate reductase (KIVR), which is the sole provider of D ‐hydroxyisovalerate (D ‐Hiv), a common precursor for cyclooligomer depsipeptide biosynthesis in Beauveria bassiana. KIVR and related hypothetical oxidoreductases encoded in fungal genomes are similar to ketopantoate reductases but not to D ‐hydroxycarboxylate dehydrogenases. We demonstrate that a KIVR knockout B. bassiana strain can be used for the efficient mutasynthesis of unnatural beauvericin congeners. Simultaneous feeding of precursor analogues enabled the combinatorial mutasynthesis of scrambled beauvericins, some assembled entirely from unnatural precursors. The effects of the introduced structural changes on the antiproliferative and cell migration inhibitory activities of these analogues were evaluated.     

Occurrence and levels of mycotoxins and their metabolites in human breast milk associated to dietary habits and other factors: A systematic literature review, 1984–2015

BackgroundMany studies around the world reported the occurrence of many mycotoxins and their metabolites in human breast milk. However the contamination by aflatoxin M1 and ochratoxin A were the most investigated by several countries.Scope and approachTo scrutinize all papers reporting quantitative data on the prevalence and the levels of mycotoxins and their metabolites in breast milk, also the circumstances of exposure.A systematic literature search in Pubmed, Science direct and Google scholar databases were performed to identify relevant studies, published in English from 1984 through May 2015.Key findings and conclusion63 studies met the inclusion criteria and assessed the occurrence of 29 mycotoxins & their metabolites in breast milk, regarding 7194 subjects of 31 countries. The maternal dietary habits, the socio demographic status of the mother, the seasonal variations and the sensitivity of the analytical method were the factors related to the high concentrations of AFM1 and OTA in breast milk.Studies where contamination exceeds maximum limits and exhibit real risk of public health were highlighted.     

食品中白僵菌素和恩镰孢菌素的污染情况及分析方法研究进展

介绍了食品中白僵菌素(beauvericin,BEA)和包括恩镰孢菌素A(enniatin A,ENA)、恩镰孢菌素A_1(enniatin A_1,ENA_1)、恩镰孢菌素B(enniatin B,ENB)、恩镰孢菌素B_1(enniatin B_1,ENB_1)在内的4种主要的恩镰孢菌素(enniatins,ENNs)的分类、毒性和分析方法,尤其是前处理方式和各方法定量限的研究进展。综述了西班牙、摩洛哥、意大利、日本等部分国家食品中BEA和4种ENNs的污染状况以及这5种毒素与其他主要真菌毒素的协同污染情况。提出了建立针对复杂食品基质中BEA和ENNs测定的高效液相色谱-串联质谱法。     

Occurrence of enniatins and beauvericin in 60 Chinese medicinal herbs

A total of 60 Chinese medicinal herbs were examined for contamination of the emerging Fusarium mycotoxins enniatins (ENNs) A, A1, B, B1 and beauvericin (BEA). The herbs under study are commonly used in China as both medicines and food. The dried samples of herbs were randomly collected from traditional Chinese medicine stores in Zhejiang province, China. Sample preparation was achieved by methanol extraction, followed by a simple membrane filtration step; no tedious clean-ups were involved. ENNs A, A1, B, B1 and BEA were analysed by the recently developed stable isotope dilution assays, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). With limits of detection ranging between 0.8 and 1.2 µg kg^–1 for the analytes under study, 25% of all analysed samples were contaminated with at least one of the ENNs and BEA. BEA was the most frequently detected toxin with a 20% incidence in all samples. The percentages of ENN-positive samples were lower: each single ENN was detected in 6.7–11.7% of all samples. Considering the total amounts of the five mycotoxins in single samples, values between 2.5 and 751 µg kg^–1 were found. The mean total amount in positive samples was 126 µg kg^–1. Regarding ginger, the frequent occurrence of ENNs and BEA in dried ginger could be confirmed in samples from Germany. However, in fresh ginger root the toxins were not detectable. This is the first report on the presence of ENNs and BEA in Chinese medicinal herbs.     

重要产毒镰刀菌合成白僵菌素和恩镰孢菌素研究进展

白僵菌素(beauvericin,BEA)和恩镰孢菌素(enniatins,ENNs)是由镰刀菌属的多种真菌产生的六酯肽类真菌毒素,该类毒素对上皮细胞、免疫细胞、卵巢细胞等具有很强的毒性作用。本文主要针对能产生BEA和ENNs的重要产毒镰刀菌的形态学特征、分子遗传学特征以及影响BEA和ENNs产生的环境条件如温度、底物等因素进行概述。重点阐述了产毒镰刀菌在两类毒素合成酶的基因水平和氨基酸水平的差异及影响产毒等主要因素,为BEA和ENNs及其产毒镰刀菌的预防和控制提供理论依据。     

不同基质中蝉花产白僵菌素研究

目的 将蝉花菌种接种于不同基质培养一定时间后,检测培养物中白僵菌素和恩镰孢菌素含量。方法 将一株蝉花菌种分别接种于4种液体培养基和4种固体培养基中,25 ℃下连续培养1～7周,采用高效液相色谱-串联质谱法测定蝉花培养物中的白僵菌素和恩镰孢菌素。结果 该株蝉花在4种液体培养基上培养不同时间后,白僵菌素的检出率在42.9%～100.0%之间,检出浓度范围为1.0～94.6 μg/L;在4种固体培养基上培养不同时间后,白僵菌素的检出率均为100.0%,检出水平范围在60.9～44 677.5 μg/kg。在4种液体和4种固体培养物中均未检出恩镰孢菌素。结论 该株蝉花在8种试验培养基上培养一定时间后均可产生白僵菌素,且在固体培养基上产生的白僵菌素水平远高于液体培养基,但在两类培养基上均不产生恩镰孢菌素。     

Effect of fungal strain and cereal substrate on in vitro mycotoxin production by Fusarium poae and Fusarium avenaceum

Single conidia strains of Fusarium poae and Fusarium avenaceum were investigated for their ability to synthesise mycotoxins in vitro. In a first experiment, rice was inoculated with three strains of F. poae and three strains of F. avenaceum. In a second experiment, the same strains were cultivated on four different cereal substrates. For both experiments, the colonised substrates were analysed using liquid chromatography coupled with tandem mass spectrometric detection (LC-MS/MS) for the content of Fusarium mycotoxins. On rice, a strong effect of strains on mycotoxin content was found. With the analyses of different cereal types, substantial substrate effects were observed. For F. poae, these effects were highly dependent on the strain. The results of this study are of benefit for risk assessment concerning naturally infected grain samples since substrate effects could also be triggered by different cereal species or varieties, and thus could lead to variable mycotoxin contamination in cereal products intended for human consumption.     

Evaluation of Beauvericin Toxicity with the Bacterial Bioluminescence Assay and the Ames Mutagenicity Bioassay

ABSTRACT: The bacterial bioluminescence assay was used to determine the acute toxicity level of beauvericin, which was found to be moderate with an EC₅₀ of 94 ± 9 μg/ml. The Ames test was used to evaluate mutagenicity of this mycotoxin. Assays were carried out on 5 Salmonella typhimurium standard tester strains. The spot test was performed on a single beauvericin concentration of 2 μg/plate. The plate incorporation test was done over a range of concentrations (0.2 to 500 μg/plate), both with and without S9. A negative control and 3 positive controls, 2-aminofluorene, sodium azide, and dexon, were included in the assays. Beauvericin was nonmutagenic to any of the standard tester strains used, either with or without metabolic activation.     

Utility of the phylotoxigenic relationships among trichothecene-producing Fusarium species for predicting their mycotoxin-producing potential

Species of the genus Fusarium are well-known plant pathogens and mycotoxigenic fusaria are associated with health hazards to humans and animals. There is a need to understand the mechanisms of mycotoxin production by Fusarium species and to predict which produce mycotoxins. In this study, the Fusarium phylogenetic tree was first inferred among trichothecene producers and related species. We reconstructed the maximum likelihood (ML) tree based on the combined data from nucleotide sequences of rDNA cluster regions, the β-tubulin gene (β-tub) and the elongation factor 1α gene (EF-1α). Second, based on this tree topology, the ancestral states of the producing potential of type A and B trichothecenes (TriA and TriB), zearalenone (ZEN), moniliformin (MON), beauvericin (BEA) and enniatins (ENN) were reconstructed using the maximum parsimony (MP) method based on the observed production by extant species as reported in the literature. Finally, the species having the potential to produce each of these six mycotoxins was predicted on the basis of the parsimonious analysis. The ML tree indicated that the Fusarium species analysed in this study could be divided into two major clades. Clade I was divided into four distinct subclades: I-a, I-b, I-c and I-d. Furthermore, the parsimony reconstruction suggested that the potential for producing MON and ZEN was gained or lost only once, and that the producing potential for TriA and TriB, BEA and ENN was repeatedly gained and lost during the evolutionary history of the Fusarium species analysed in this study. Interestingly, the results showed the possibility that several species, about which reports were scarce with regard to mycotoxin production, have the potential to produce one or more of the six evaluated in this study. The phylogenetic information therefore helps one to predict the mycotoxin-producing potential by Fusarium species, and these “phylotoxigenic relationships” may be useful for predicting the pathogenicity of fungi.     

Fungal and bacterial metabolites associated with natural contamination of locally processed rice (Oryza sativa L.) in Nigeria

This study reports the fungal and bacterial metabolites associated with natural contamination of 38 composite samples of locally processed rice from five agro-ecological zones of Nigeria (AEZs). The samples were evaluated for the presence of microbial metabolites by LC-MS/MS. Among the identified metabolites, 63 fungal and 5 bacterial metabolites were measured at varying concentrations and occurrence levels. Fusarium toxins had the highest incidence of 79%, but occurred in low amounts with fumonisin B₁ (FB₁) having the highest percentage incidence of 39.5% and a mean of 18.5 µg/kg. Among the Aspergillus toxins, aflatoxins (AFs) occurred in 36.9% of the rice samples, with aflatoxin B₁ (AFB₁) having the highest occurrence level of 18.4% and a mean value of 5 µg/kg. About 12 metabolites had incidence levels > 50%, including beauvericin (BEA) and tryptophol, which had occurrence levels of 100%. Among the emerging toxins under evaluation by international organisations such as the European Food Safety Authority (EFSA) and the Food and Agriculture Organisation of the United Nations (FAO), citrinin, sterigmatocystin (STER) and beauvericin were detected with maximum values of 207, 125 and 131 μg/kg, respectively. This paper also reports the first documented evidence of the contamination of Nigerian rice by bacterial and Alternaria metabolites, nivalenol, kojic acid, STER, moniliformin, fusaric acid, fumonisin B₃, citrinin, 3-nitropropionic acid, andrastin A, cytochalasins, emodin and physicon.