自溶霉素生物合成的调控及后修饰机制研究

Autolytimycin, a novel compound belonging to the group of ansamycins, was discovered from a new species of actinomycetes called Streptomyces autolyticus. Autolytimycin possesses potent anti-tumor activity. However, the low yield of autolytimycin severely hampers its further development. Since the total synthesis of autolytimycin by chemical methods is very difficult, the key to improve the production of autolytimycin is to modify its biosynthesis through genetic engineering, on the basis of a better understanding of the mechanism. In our previous work, the gene culster responsible for the biosynthesis of autolytimycin was cloned and sequenced, and the genes involved were identified. In this project, the function of key regulatory genes and tailoring genes that are involved in autolytimycin biosynthesis will be studied to understand the mechanism of its biosynthesis, especially the factors that affect its yield. The genes affecting the yield of autolytimycin will be genetically manipulated to increase its production, which would set up the foundation for further development of autolytimycin as an anti-tumor drug.

自溶霉素是我们从放线菌新种自溶链霉菌中发现的一个新的安莎霉素类化合物,具有很强的抗肿瘤活性。然而，自溶霉素的产率较低，严重阻碍了对它的进一步研发。由于自溶霉素的化学合成极为困难，因而对其生物合成进行研究和遗传改良就成为提高自溶霉素产率的关键所在。前期工作中我们克隆和测序了自溶霉素生物合成的全基因簇，确定了与其生物合成有关的基因。本项目拟对自溶霉素生物合成途径中的关键调控基因和后修饰酶基因的功能进行更加深入的研究，了解自溶霉素生物合成的调控及后修饰机制，探明影响自溶霉素产率的关键因素，然后对相关基因实施遗传工程改造以提高自溶霉素产率，为进一步研发此类抗肿瘤新药奠定基础。

提高新型抗肿瘤化合物自溶霉素产量并获得更加高效、低毒的新结构类似物是这类活性化合物能够得到实际应用的关键。在本项目中，我们对自溶链霉菌中参与自溶霉素生物合成的关键基因的功能及其调控机制进行了研究，并通过遗传改造有效提高了自溶霉素的产率。具体研究内容包括：构建了调控基因almRI、almRII和almRIII的敲除突变株，通过对其发酵产物的检测初步证明了这些基因在自溶霉素生物合成中起正调控作用；使用实时定量PCR检测了上述调控基因在不同条件下的表达，发现其与自溶霉素产量之间存在正相关性；通过RT-PCR和转录组测序对相关基因的表达进行了分析，证明自溶霉素生物合成基因簇中多个基因的表达受到这三个调控基因的控制；表达纯化了上述基因编码的蛋白并对其作用对象进行了研究，部分确定了它们的作用靶目标及作用位点；构建并分析了后修饰酶基因almM的敲除突变株，证明其在此类化合物苯酚环的氧化过程中起作用；使用实时定量PCR检测了almM基因在不同发酵阶段的表达，发现其与自溶霉素产量之间存在正相关性；表达纯化了AlmM蛋白并对其酶学特性进行了研究；优化了发酵条件，从所构建almM基因敲除突变株中分离纯化了足够的自溶霉素样品。本项目的实施可以帮助我们进一步认识链霉菌中复杂而精巧的次级代谢过程，也为自溶霉素的进一步研发奠定了基础。