橙色红曲菌As3.4384 orf7基因缺失株的构建及其功能分析

Construction of the Monascus aurantiacus As3.4384 orf7 Gene Deletion Strains and Functional Analysis

红曲菌能产生多种有益的次级代谢产物,但红曲菌也产生一种对人和哺乳动物肝和肾有毒害的毒素,即桔霉素。因此控制毒素的产生是保障红曲产品安全性所必须的。故对桔霉素的合成途径及相关的基因做深入了解。6个桔霉素合成相关的基因成簇位于21 kb的DNA片段上。克隆了一个新基因(orf7基因),其位于该基因簇的外侧。采用基因敲除技术,构建红曲菌orf7基因缺失菌株。并采用紫外分光光度法检测orf7基因缺失菌株的红曲色素产量,HPLC法检测其桔霉素产量。orf7缺失菌株产红曲色素能力与出发菌株As3.4384相比没有变化;产桔霉素培养13～19 d,桔霉素的产量与出发菌株As3.4384相比增加了142.4%。从而证实orf7基因与桔霉素代谢相关。

Monascus can produce several kinds of produce citrinin that is harmful to mammals because of beneficial secondary metabolites. But Monascus can also nephrotoxic and hepatoxic activity. Thus it is essential to control the production of this toxin in order to increase the safety of Monascus-related product. Extensive research on citrinin pathway and genes involving in this toxin biosynthesis is conducted. Six genes responsible for citrinin biosynthesis are clusted within 21kb DNA region. A gene（orf7） which locates in the vicinity of orf5 was cloned. The orf7 gene disruption mutants in M. aurantiacus As3.4384 were constructed. The citrinin yield of the orf7 gene disruption mutants was analyzed by HPLC and the pigments were analyzed by UV spectrophotometer. From 13th to 19th day of cultivation, the yield of citrinin of orf7 disruptant increased 142.4% compared to wild-type As3.4384. So it was confirmed that orf7 was related to metabolism of citrinin.