| 王红,殷欣,孟庆洲,等.基因组信息指导下海参共附生Penicillium sclerotiorum SD-36嗜氮酮类化合物的挖掘[J].中国海洋药物,2022,41(4):1-11. |
| 基因组信息指导下海参共附生Penicillium sclerotiorum SD-36嗜氮酮类化合物的挖掘 |
| Mining of azaphilones from a sea cucumber epiphytic fungus Penicillium sclerotiorum SD-36 under the guidance of genomic information |
| 投稿时间：2021-07-15 修订日期：2021-09-30 |
| DOI： |
| 中文关键词: Penicillium sclerotiorum SD-36 双PKS基因簇 转录因子过表达 isochromophilone VI sclerotiorin C |
| English Keywords:Penicillium sclerotiorum SD-36 double PKS gene cluster overexpression of transcription factor isochromophilone VI sclerotiorin C |
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| 中文摘要: |
| 目的 结合生物信息学与分子遗传操作技术深入挖掘海参共附生菌核青霉(Penicillium sclerotiorum)SD-36的活性次级代谢产物,并探析其生物合成基因簇中转录调控因子的作用。方法 基于本课题组前期P. sclerotiorum SD-36的基因组信息,利用antiSMASH预测了一条具有合成嗜氮酮类化合物潜力的双PKS基因簇。针对该簇中一个锌指转录因子PsAza1构建了含潮霉素抗性基因和强启动子pgpdA的转录因子基因过表达盒,转化P. sclerotiorum SD-36原生质体后,经过抗性筛选及PCR验证获得阳性转化子OE::PsAza1。发酵培养后HPLC检测次级代谢产物变化并通过qRT-PCR验证基因簇核心基因的转录水平。结果 OE::PsAza1的多种次级代谢产物产量增加,其中两个化合物通过质谱、核磁数据鉴定为活性嗜氮酮类isochromophilone VI和sclerotiorin C,其产量较野生型分别增加了约6倍和4.5倍。qRT-PCR检测该簇中两个聚酮合酶基因及Psaza1基因的转录水平,显示上调60-80倍。结论 首次明确P. sclerotiorum SD-36的双PKS基因簇编码活性嗜氮酮类化合物,且转录因子PsAza1正向调控该簇核心基因的表达水平及化合物产量。本研究结果为P. sclerotiorum SD-36中化合物isochromophilone VI和sclerotiorin C的生物制备及调控研究奠定理论基础。 |
| English Summary: |
| Objective Combined with bioinformatics and molecular genetic manipulation techniques, the active secondary metabolites of a sea cucumber epiphytic fungus Penicillium sclerotiorum SD-36 were mined, and the role of transcriptional regulatory factors in their biosynthetic gene cluster was analyzed. Methods Based on the previous genomic information of P. sclerotiorum SD-36, antiSMASH platform was used to predict and annotate a double PKS gene cluster with the potential to synthesize azaphilones. A gene overexpression box of transcription factor PsAza1, containing hygromycin resistance gene and strong promoter pgpdA, was constructed. After transforming the gene overexpression box into the protoplast of P. sclerotiorum SD-36, a positive transformant OE::PsAza1 (Overexpression::PsAza1) was obtained through resistance screening and PCR verification. The yeild of secondary metabolites of OE::PsAza1 after fermentation were detected by HPLC and the transcriptional levels of core genes in the gene cluster were verified by qRT-PCR. Results The yeild of several secondary metabolites of OE::PsAza1 were increased. Among them, two compounds were identified as active azaphilones isochromophilone VI and sclerotiorin C by mass spectrometry and NMR data, which were about 6-fold and 4.5-fold higher than that of wild type, respectively. The transcriptional levels of two polyketone synthase genes and PsAza1 gene in this cluster were detected by qRT-PCR, and the results showed that they were up-regulated by 60-80 times. Conclusion For the first time, the double PKS gene cluster of P. sclerotiorum SD-36 encoded active azaphilones, and the transcription factor PsAza1 positively regulated the expression level of the core gene in the cluster and compound production. The results of this study laid a theoretical foundation for the biological preparation and regulation of isochromophilone VI and sclerotiorin C from P. sclerotiorum SD-36. |
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