Abstract：
Aspergillus flavus can colonize important food staples and produce aflatoxins, a group of toxic and carcinogenic secondary metabolites. Previous in silico analysis of the A. flavus genome revealed 56 gene clusters predicted to be involved in the biosynthesis of secondary metabolites. A. flavus secondary metabolites produced during infection of maize seed are of particular interest, especially with respect to their roles in the biology of the fungus. A predicted nonribosomal peptide synthetase-like (NRPS-like) gene, designated asaC (AFLA_023020), present in the uncharacterized A. flavus secondary metabolite gene cluster 11 was previously shown to be expressed during the earliest stages of maize kernel infection. Cluster 11 is composed of six additional genes encoding a number of putative decorating enzymes as well as a transporter and transcription factor. We generated knock-out mutants of the seven predicted cluster 11 genes. LC-MS analysis of extracts from knockout mutants of these genes showed that they were responsible for the synthesis of the previously characterized antimicrobial mycotoxin aspergillic acid. Extracts of the asaC mutant showed no production of aspergillic acid or its precursors. Knockout of the cluster 11 P450 oxidoreductase afforded a pyrazinone metabolite, the aspergillic acid precursor deoxyaspergillic acid. The formation of hydroxyaspergillic acid was abolished in a desaturase/hydroxylase mutant. The hydroxamic acid functional group in aspergillic acid allows the molecule to bind to iron resulting in the production of a red pigment in A. flavus identified previously as ferriaspergillin. A reduction of aflatoxin B1 and cyclopiazonic acid that correlated with reduced fungal growth was observed in maize kernel infection assays when aspergillic acid biosynthesis in A. flavus is halted.
摘要：
黄曲霉可以定殖重要的主食并产生黄曲霉毒素，一组有毒和致癌的次级代谢产物。先前对黄曲霉基因组的计算机模拟分析显示，有56个基因簇被预测参与次级代谢产物的生物合成。在玉米种子感染过程中产生的黄质次生代谢产物特别令人感兴趣，特别是它们在真菌生物学中的作用。预测的非核糖体肽合成酶样（NRPS样）基因，指定为asaC(AFLA_023020)，存在于未表征的黄曲霉次级代谢物基因簇11中，先前已显示在玉米籽粒感染的最早阶段表达。簇11由六个额外的基因组成，这些基因编码许多推定的装饰酶以及转运蛋白和转录因子。我们产生了7个预测的簇11基因的敲除突变体。这些基因的敲除突变体的提取物的LC-MS分析表明，它们负责合成先前表征的抗微生物霉菌毒素曲霉酸。asaC突变体的提取物显示没有产生曲霉酸或其前体。敲除簇11P450氧化还原酶提供了吡嗪酮代谢物，曲霉酸前体脱氧曲霉酸。去饱和酶/羟化酶突变体中羟基曲霉酸的形成被废除。曲霉酸中的异羟肟酸官能团使分子与铁结合，从而在先前鉴定为费拉西林的黄曲霉中产生红色颜料。当黄曲霉中的曲霉酸生物合成停止时，在玉米籽粒感染测定中观察到与真菌生长减少相关的黄曲霉毒素B1和环吡唑酸的减少。
