Transposon-based identification of a negative regulator for the antibiotic hyper-production in Streptomyces
Transposon-based identification of a negative regulator for the antibiotic hyper-production in Streptomyces
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基于转座子的链霉菌抗生素过量产生负调节因子的鉴定
DOI：
10.1007/s00253-018-9103-5
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发表时间：
2018-08-01
影响因子：
5
通讯作者：
Li, Yong-Quan
中科院分区：
文献类型：
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作者：
Luo, Shuai;Chen, Xin-Ai;Li, Yong-Quan
链霉菌次级代谢产物的产生受到一个复杂的调控网络的精确、精细和分级调控。一些高价值次级代谢产物产量低的主要原因之一是其生物合成基因簇的表达受到抑制，据推测是由一些基因簇外的负调控因子引起的。鉴定这些阻遏物并根据其调控机制消除其抑制作用将是提高其产量的有效途径。为了证明这一概念，使用来自玫瑰孢链霉菌的抗生素达托霉素，我们引入了基于Himar 1的随机诱变，并结合细菌引导的筛选策略来鉴定转录调节因子PhaR，其功能缺失导致基因簇表达增加约2.68倍，并且在烧瓶发酵或在补料发酵中达托霉素产量增加约6.14倍或43%。分批发酵。进一步的研究表明PhaR通过与达托霉素生物合成基因簇的启动子（dptEp）直接结合，负调控达托霉素生物合成基因簇的表达。此外，phaR表达在发酵过程中逐渐下降，并且PhaR通过直接结合其自身启动子而被正向自调节，这导致正反馈调节以持续降低phaR表达。同时，下降的PhaR蛋白消除了其在达托霉素生产过程中的抑制作用。所有这些结果都支持我们的策略将成为提高抗生素产量的基因筛选和合理工程的有力方法，并有可能广泛应用于其他链霉菌物种。
Production of secondary metabolites in Streptomyces is regulated by a complex regulatory network precisely, elaborately, and hierarchically. One of the main reasons for the low yields of some high-value secondary metabolites is the repressed expression of their biosynthetic gene clusters, supposedly by some gene cluster out-situated negative regulators. Identification of these repressors and removal of the inhibitory effects based on the regulatory mechanisms will be an effective way to improve their yields. For proof of the concept, using an antibiotic daptomycin from Streptomyces roseosporus, we introduced Himar1-based random mutagenesis combined with a reporter-guided screening strategy to identify a transcriptional regulator PhaR, whose loss-of-function deletion led to about 2.68-fold increase of the gene cluster expression and approximately 6.14-fold or 43% increased daptomycin production in the flask fermentation or in the fed-batch fermentation, respectively. Further study showed that PhaR negatively regulates the expression of daptomycin biosynthetic gene cluster by direct binding to its promoter (dptEp). Moreover, phaR expression gradually drops down during fermentation, and PhaR is positively auto-regulated by directly binding to its own promoter, which results in positive feedback regulation to persistently reduce phaR expression. Meanwhile, the declining PhaR protein remove its repressive effects during daptomycin production. All these results support that our strategy would be a powerful method for genetic screening and rational engineering for the yield improvement of antibiotics, and could be potentially used widely in other Streptomyces species.