ABC转运蛋白基因slnTI和slnTII与盐霉素生物合成的相关性

Involvement of ABC transporter genes slnTI and slnTII in salinomycin biosynthesis

摘要:

【目的】slnTI和slnTII是盐霉素生物合成基因簇中可能的两个转运蛋白基因，根据生物信息学的分析推测它们属于ABC转运蛋白家族。其中，slnTI编码ABC转运蛋白的ATP结合亚基，slnTII编码ABC转运蛋白的跨膜亚基，推测它们可能与盐霉素的外排有关。通过slnTI和slnTII的基因中断与超量表达研究它们对盐霉素生物合成产量和抗性的影响。【方法】利用REDIRECT?技术，在盐霉素产生菌白色链霉菌XM211中分别构建了slnTI和slnTII的基因置换突变株LJ01和LJ02，并通过基因回补对突变株进行了验证。利用整合型表达载体pPM927在白色链霉菌XM211中对slnTI和slnTII进行串联超量表达。将slnTI和slnTII导入变铅青链霉菌1326中进行异源表达，通过液体培养实验检测衍生菌株对盐霉素的抗性。【结果】相比出发菌株XM211，突变株LJ01中盐霉素的产量下降了27.2%，LJ02下降了45.4%，LJ01和LJ02中结构基因slnA3和调控基因slnR的转录水平都有明显降低。超量表达菌株LJ03中盐霉素的产量提高了14.6%，转录结果显示LJ03中不仅slnTI和slnTII自身转录水平有大幅提高，而且slnA3和slnR转录水平也显著升高。抗性检测结果表明，异源表达菌株变铅青链霉菌LJ04对盐霉素的抗性水平略有提高。【结论】slnTI和slnTII是与盐霉素生物合成和外排有关的ABC转运蛋白基因，但并不是白色链霉菌XM211对盐霉素的主要抗性基因。

Abstract:

[Objective] In the salinomycin biosynthetic gene cluster, slnTI and slnTII encode the ABC transporter ATP-binding subunit and trans-membrane subunit, respectively. We speculate that slnTI and slnTII are most likely related to salinomycin export. Here, the role of slnTI and slnTII on salinomycin biosynthesis and resistance was investigated by gene replacement and overexpression. [Methods] Using REDIRECT? technology, the slnTI mutant LJ01 and the slnTII mutant LJ02 were constructed, and further verified by trans-complementation with cloned corresponding genes. SlnTI and SlnTII were overexpressed in the wild-type Streptomyces albus XM211. Also, slnTI and slnTII were introduced into S. lividans 1326, and the resistance to salinomycin of the resulted strains was tested. [Results] Compared with the wild-type strain, salinomycin production in LJ01 and LJ02 decreased by 27.2% and 45.4%, respectively. The transcription of structure gene slnA3 and regulatory gene slnR were both reduced in LJ01 and LJ02. The overexpression of slnTI and slnTII in the wild-type XM211 resulted in a 14.6% enhanced salinomycin production. Correspondingly, the transcription of slnA3 and slnR was also increased. Additionally, heterologous expression of slnTI and slnTII in S. lividans 1326 slightly increased its resistance to salinomycin. [Conclusion] slnTI and slnTII are identified to be related to the production of salinomycin, but not to be the main resistant gene for salinomycin in the producer.