链霉菌多效调控子—WblA激活隐性抗生素合成基因簇 (tyl)的分子机制

Streptomycetes, as the main source of antibiotics, have been intensively exploited for discovering new drug candidates to combat the evolving pathogens. With the quick development of genomic DNA sequencing, we will have good opportunity to search more novel antibiotics. wblA, encoding a pleiotropic regulatory protein, was discovered and cloned in Streptomyces ansochromogenes 7100. wblA disruption mutant of Streptomyces ansochromogenes 7100 (ΔwblA) was constructed by homologous recombination. ΔwblA failed ability to form spores and produce nikkomycin, a natural product of Streptomyces ansochromogenes 7100 (wild-type strain). Antibacterial activity against Staphylococcus aureus and Streptococcus pneumonia was observed with fermentation broth of ΔwblA but not with that of the wild-type strain. The compound (named as Tylosin Derivant, TylD) produced by ΔwblA was characterized as 16-membered macrolides by mass spectrometry and nuclear magnetic resonance spectroscopy. According to the genome analysis, the biosynthetic gene cluster of TylD was cloned and identified. Base on previous work, this project is to study two regulatory genes situated in tyl gene cluster (wblA is not situated in tyl), meanwhile to search and identify the target genes recognized by WblA using ChIP-seq/RNA-seq, and to reveal molecular mechanism of WblA ( a pleiotropic regulator) activating tyl cryptic-antibiotic biosynthetic gene cluster in Streptomyces ansochromogenes. All of the studies mentioned above are aimed to discover novel regulatory mechanism and novel antibiotcs.

wblA是放线菌特有的基因，在发育分化和抗生素生物合成中发挥了重要作用，本实验室通过同源双交换获得wblA突变株(ΔwblA)，wblA敲除导致圈卷产色链霉菌不能正常发育分化, 同时尼可霉素也不能被合成。wblA阻断突变株的发酵液显示了抑制金黄色葡萄球菌和肺炎链球菌的活性，而这种活性是野生型菌株发酵液所不具有的。通过HPLC、质谱和核磁共振解析了化合物的结构，将其命名为TylD (Tylosin Derivant)，并得到了TylD的生物合成基因簇(tyl)。本申请项目在原工作基础上拟对基因簇中调控基因(tylR1和tylR2)的功能进行研究、通过ChIP-Seq，并结合RNA-seq和EMSA等实验寻找WblA调控蛋白所识别的靶基因， 阐明WblA激活tyl隐性基因簇的分子机制，发现由多效调控子介导抗生素生物合成的新调控机制，为更多隐性基因簇的激活和得到新型的抗生素提供重要的依据。

随着抗生素乱用和过量使用引起了诸多抗药性菌株的不断上升，人类正面临着全球性的健康危机。链霉菌基因组测序结果揭示大量次级代谢生物合成基因簇的产物是未知的，有必要建立不同的方法去激活这些隐性途径而得到有重要价值的生物活性产物。我们通过圈卷产色链霉菌野生型及其敲除株∆wblA的RNA-seq数据进行了转录组分析，结果表明一些重要的差异表达基因与尼可霉素生物合成阻断和泰乐菌素衍生物的产生有密切关系，尤其是两个编码RsbR的差异表达基因（san7324和san7324L）的敲除可以使oviedomycin产生。这些结果为新型候选药物的发现和隐性生物合成基因簇的激活提供了重要的思路。为了阐明WblA作用的分子机制，我们通过纯化WblA和其同源蛋白WhiB4，EMSA实验显示在还原状态下WhiB4能结合本身编码基因的启动子PwhiB4，而WblA不能结合本身编码基因的启动子PwblA，推测WblA行使功能的方式与WhiB4是不同的，我们进一步通过细菌单杂交 (bacterial one-hybrid, B1H）技术，检测了WblA与其靶基因的结合能力，发现WblA可以通过直接抑制化合物1，2，3生物合成基因簇中调控基因（tylR1和tylR2）及结构基因（tyl7*）的转录，使该基因簇保持沉默状态。本研究初步阐明了WblA调控的分子机制、为激活更多天然活性产物生物合成基因簇的表达奠定了重要基础。