黑曲霉中生物合成赭曲霉毒素A的非核糖体肽合成酶基因的鉴定

赭曲霉毒素A(ochratoxin A,OTA)是国际癌症研究机构认定的"2B"类致癌物。黑曲霉Aspergillus niger是美国食品药品监督局认可的食品安全菌。然而近年来陆续发现某些黑曲霉菌株能够产生OTA,这会对人类健康构成潜在威胁。阐明黑曲霉生物合成OTA的关键基因有助于理解OTA生物合成机制,这对OTA污染的防控具有重要意义。本研究克隆了产OTA黑曲霉中非核糖体肽合成酶(NRPS)编码基因(An15g07910),并对其进行了生物信息学分析,在此基础上采用同源重组的方法敲除了该基因,获得了一株性能稳定的敲除突变株Δnrps。与野生株相比,Δnrps突变株的表型在CYA培养基中并无明显改变,但在7d培养期间完全失去了合成赭曲霉毒素α(ochratoxinα,OTα)和OTA的能力,而赭曲霉毒素β(ochratoxinβ,OTβ)的合成不受影响。在野生株培养过程中,该nrps基因前4d表达量逐渐增大,并在第4天达到最高,随后基因表达量逐渐下降并趋于稳定,这与OTA的含量变化基本一致。结果表明该nrps基因(An15g07910)参与OTA的生物合成,其编码的NRPS可能负责催化苯丙氨酸部分和二氢异香豆素部分的交联。

Identification of a nonribosomal peptide synthetase gene involved in the biosynthesis of ochratoxin A in Aspergillus niger

Ochratoxin A (OTA) is classified as a renal carcinogen of animals and a possible (group 2B) human carcinogen by the International Agency for Research on Cancer. Aspergillus niger has been generally recognized as safety (GRAS) by the US Food and Drug Administration (FDA). However, there have been increasingly reported that some isolates of A. niger are able to produce OTA, arousing people’s great concern. Elucidating the key genes required for the biosynthesis of OTA is helpful to understand the mechanism of OTA production in A. niger, which is of great significance for the development of effective preventive and control strategies to reduce the risk of OTA contamination in foods and other products. In this study, the non-ribosomal peptide synthase (NRPS) coding gene (An15g07910) of A. niger was cloned and analyzed by bioinformatics. The gene was knocked out by homologous recombination, and a stable knockout mutant, Δnrps, was obtained. No statistically significant differences in growth, colony morphology and conidial morphology were observed in the Δnrps mutant in comparison with the wild-type strain of A. niger on the Czapek yeast autolysate (CYA) agar plates. Disruption of the gene An15g07910 completely eliminated the production of ochratoxin α (OTα) and OTA during 7 days of culture, but it does not affect the production of ochratoxin β (OTβ). The highest expression level of the gene An15g07910 was detected on day 4 after inoculation, and it decreased and tended to be stable afterward, being basically consistent with the change of OTA content. The results showed that the nrps gene (An15g07910) was involved in OTA biosynthesis, and its encoded NRPS may be responsible for the cross-linking of phenylalanine and dihydroisocoumarin sectors.