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首页> 外文期刊>Applied Microbiology >Identification of a Halogenase Involved in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius
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Identification of a Halogenase Involved in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius

机译：鉴定Aspergillus Carbonarius中涉及核苷酸生物合成的卤素酶

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Aspergillus carbonarius is the main responsible fungus of ochratoxin A (OTA) contamination of grapes and derived products. To date, the biosynthetic mechanism of this mycotoxin has been partially elucidated. Availability of genome sequence of A. carbonarius has allowed the identification of a putative gene cluster involved in OTA biosynthesis. This region hosts the previously characterized AcOTAnrps and AcOTApks genes encoding two key enzymes of the biosynthetic pathway. At about 4,400 nucleotides downstream of these loci, a gene encoding a putative flavin dependent-halogenase came out from the annotation data. Its proximity to OTA biosynthetic genes and its sequence analysis have suggested a role in the biosynthesis of OTA, directed to the introduction of the chlorine atom in the C-5 position of the final molecular structure of this mycotoxin. The deduced protein sequence of the halogenase gene, we designated AcOTAhal , shows a high similarity to a halogenase that is located in the OTA cluster of A. niger . The deletion of the halogenase gene completely eliminated the production of ochratoxin A in A. carbonarius and determined a significant increase of ochratoxin B, as confirmed by mass spectrometry analysis. Moreover, its expression profile was similar to the two biosynthetic genes previously identified, AcOTApks and AcOTAnrps , indicating a strong correlation of the AcOTAhal gene with the kinetics of OTA accumulation in A. carbonarius. Therefore, experimental evidence confirmed that the chlorination step which converts OTB in OTA represents the final stage of the biosynthetic pathway, supporting our earlier hypothesis on the order of enzymatic steps of OTA biosynthesis in A. carbonarius .IMPORTANCE Ochratoxin A is a potent mycotoxin classified as a possible carcinogen for humans, and Aspergillus carbonarius is the main agent responsible for OTA accumulation in grapes. We demonstrate here that a flavin-halogenase is implicated in the biosynthesis of OTA in A. carbonarius . The encoding gene, AcOTAhal , is contiguous to biosynthetic genes that we have already described ( nrps and pks ), resulting as part of the biosynthetic cluster. The encoded protein is responsible of the introduction of chlorine atom in the final molecular structure and acts at the last step in the pathway. This study can be considered a continuation of an earlier study wherein we started to clarify the molecular basis of OTA biosynthesis in A. carbonarius , which has not been completely elucidated until now. This research represents an important step forward to a better understanding of the production mechanism, which will contribute to the development of improved control strategies to reduce the risk of OTA contamination in food products.
机译：曲霉属Carbonarius是Ochratoxin A（OTA）葡萄和衍生产品的主要负责任真菌。迄今为止，这种霉菌毒素的生物合成机制已被部分阐明。 A. Carbonarius的基因组序列的可用性允许鉴定参与OTA生物合成的推定基因簇。该区域宿主以先前其特征的Acotanrps和Chotapks基因编码，编码了生物合成途径的两个关键酶。在这些基因座下游的约4,400个核苷酸，从注释数据中，编码推定的黄素依赖性 - 卤素酶的基因出来。它邻近OTA生物合成基因及其序列分析表明在OTA的生物合成中的作用，指导在该霉毒素的最终分子结构的C-5位置引入氯原子。我们指定Acotahal的卤素酶基因的推导蛋白质序列显示出与位于A.尼日尔的OTA簇中的卤素酶的高相似性。通过质谱分析证实，完全消除了卤素酶基因的缺失，并确定了核毒素B的显着增加，并确定了通过质谱分析证实的Ochratoxin B.此外，其表达谱类似于先前鉴定的两种生物合成基因，表达Acotapks和AcotanRPS，表明Acotahal基因与A. Carbonarius中的OTA积累动力学的强烈相关性。因此，实验证据证实，转化OTA中OTB的氯化步骤代表了生物合成途径的最终阶段，支持我们在A. Carbonarius中ota生物合成的酶促步骤的酶促步骤的序列中的前阶段。分析ochratoxin a是一种典故的霉菌毒素用于人类的可能的致癌物质，曲霉属Carbonarius是负责葡萄中的OTA积累的主要试剂。我们在此证明，Flavin-卤素酶涉及A. Carbonarius中OTA的生物合成。编码基因，Acotahal，与我们已经描述的生物合成基因邻接（NRPS和PKS），导致作为生物合成簇的一部分。编码蛋白质负责在最终分子结构中引入氯原子，并在途径的最后一步中作用。该研究可以被认为是早期研究的延续，其中我们开始澄清OTA生物合成中的分子基础在A. Carbonarius中，其直到现在尚未完全阐明。本研究代表了更好地理解生产机制的重要一步，这将有助于发展改善的控制策略，以降低食品中OTA污染的风险。

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