基于转录组学技术研究真菌诱导子促进纳他霉素生物合成的机制

Natamycin (Pimaricin) is a commercially important polyene antifungal compound and was approved by many countries to be as a natural product food preservative in many food industries.Fermentation is the mainly way to produce natamycin. But,the way have some shortcoming, for example, low production and slow industrialization. Some paper had proved that adding fungal elicitor was an effective method to improve the prodction of natamycin,but the elicitor and the mechanism of inducing are not clear. In this study, the aim is mainly to study the inducing mechanism why the elicitor produced by Penicillium chrysogenum can enhance the yield of natamycin produced by Streptomyces natalensis. This research is firstly to identify the structure of elicitor using the advanced isolation techniques and spectrum methods. The latter is that the RNA-Seq technique was employed to compare the gene expression profilings of the strain which is induced by the fungal elicitors and the control which is not induced by the fungal elicitors, and to screen the specific related genes with inducing. Then, temporal and spatial expression regulations of the related genes were investigated by RT-qPCR. Full length cDNAs of the related genes were cloned analysized and verified. So, the inducing mechanism is clarified by the transcriptomics of Streptomyces natalensis. The strategies and conclusions obtained from the study are not only the academic references for the study of natamycin and other natural products, will but also promote the application in food field.

纳他霉素是已被大多数国家批准在食品中使用的能抑制真菌生长的一种生物防腐剂，发酵法是其最主要的生产方式。由于发酵法产量较低，导致价格居高不下，严重阻碍了其推广应用。此前研究表明，添加某些真菌诱导子是提高纳他霉素产量的有效方式，但对真菌诱导子及其诱导机制缺乏研究。本课题以产黄青霉作为诱导子产生菌，对真菌诱导子中关键诱导成分和诱导机理进行研究。前者采用活性跟踪方法和活性成分消减技术，结合现代分离、鉴定方法对诱导子进行结构鉴定。后者应用高通量RNA-Seq测序技术，从转录水平对比添加和不加诱导子的纳塔尔链霉菌的基因表达差异，筛选出与纳他霉素合成相关的关键表达差异基因，利用RT-qPCR技术验证该差异基因的时间表达特性，克隆该基因并进行结构功能解析和功能的验证，阐明真菌诱导子的诱导机制。该研究结果不仅为真菌诱导子调节纳他霉素及其它天然产物生产提供重要理论依据，而且大大推动纳他霉素在食品领域广泛应用。

纳他霉素是一种已在食品中广泛使用的生物防腐剂，通过添加真菌诱导子提高其发酵产量是一种有效的方式，但其诱导机制未知，因此，揭示真菌诱导子的诱导机制具有重要的科学意义。本研究以产黄青霉作为真菌诱导子的生产菌，采用诱导活性跟踪和活性成分消减技术对真菌诱导子中关键诱导成分进行结构鉴定；应用RNA-Seq测序技术，从转录水平研究真菌诱导子促进纳他霉素生物合成的机制。研究结果如下：（1）通过分离纯化和制备，获得目标物质的UV，IR，HRMS 和 NMR等波谱学数据，鉴定出真菌诱导子为2-氨基-N-（1-（2-氨丙基）-4-氧-1,2-二氮杂环丁烷-3-基）-2-（4-对羟基苯）乙酰胺。（2）真菌诱导子可以抑制S. natalensis HW-2菌的生长、促进还原糖的利用，导致菌落和菌丝特征发生明显变化，引起胞内钙离子和活性氧水平分别提高了32.8% 100%，使PC酶和PEPC酶的活性分别提高了27.8%和 11.9%，而CS酶活性降低23.1%，从而促进前体物质草酰乙酸的积累。（3）转录组数据分析显示真菌诱导子对对数末期S.natalensis HW-2的转录水平影响最大，KEGG和Pathway分析表明影响最大的为氨基酸的合成和代谢途径，特别是分支氨基酸。（4）构建过表达载体pIB139-ilvB，对ilvB基因功能进行验证， 经发酵性能检测，ilvB 基因过表达的菌株S. natalensis HW-2-ilvB 纳他霉素产量比原始菌株提高了59.2%。本项目的顺利实施，为真菌诱导子调节纳他霉素及其它微生物次级代谢产物的生产提供重要理论依据。

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