Ansacarbamitocin生物合成机理解析及安丝菌素的定向改造

Ansacarbamitocin and ansamitocin belong to maytansinoid and are highly potent against variou tumor cell lines. The conjugants of ansamitocin and tumor-specific antibodies Kadcyla has excellent clinical effect again breast cancer. We have established the fermentation, purification, and analysis methods for ansacarbamitocin produced by Amycolatopsis sp. CP2808. The genomic library had been constructed and the corresponding gene cluster had been cloned. Complete sequencing of the biosynthetic gene cluster identifed severy unqiue enzymes with probable noval activities, which are responsible for the mutiple modifications occurred on ansarcarbamitocins. Due to the similarity and difference between ansacarbamitocins and ansamitocins, we are going to firstly elucidate the biosynthetic mechanism of ansarbamitocins, esp. the enzymatic features and orders of the post-PKS modifications. Moreover, based on the biosynthetic mechanism of ansarcabamitocin and the methodology of synthetic biology, the ansamitocin will be specifically modified with enzymes from the ansacarbamitocin biosynthetic pathway, which will generate various new derivatives for further conjugation with antibody and subsequent antitumor activity evaluation.

Ansacarbamitocins和安丝菌素均属于美登素类抗生素，对多种肿瘤细胞系都有着极强的抑制和杀灭活性。将安丝菌素与肿瘤表面的抗体进行耦联所形成的抗体药物Kadcyla在治疗乳腺癌方面表现出了非常好的治疗效果。我们成功的建立了拟无枝菌酸菌CP2808中ansacarbamitocins的发酵和检测方法，同时还构建了该菌株的基因组文库，并成功克隆了ansacarbamitocins的生物合成基因簇，发现了该基因簇中含有多个特有的新颖酶催化元件。 本项目拟以ansacarbamitocins生物合成机理的研究为切入点，开展相关后修饰基因的功能验证及相关酶学特征的研究，采用与安丝菌素比较研究的策略阐述ansacarbamitocins生物合成的后修饰机理和顺序，并利用合成生物学的相关技术，对安丝菌素分子结构进行重新设计，实现与抗体直接耦联的目的，并为药物研发提供资源。

Ansacarbamitocins和安丝菌素均属于美登素类抗生素，对多种肿瘤细胞系都有着极强的抑制和杀灭活性。将安丝菌素与肿瘤表面的抗体进行耦联所形成的抗体药物Kadcyla在治疗乳腺癌方面表现出了非常好的治疗效果。本项目拟以ansacarbamitocins生物合成机理的研究为切入点，开展相关后修饰基因的功能验证及相关酶学特征的研究，采用与安丝菌素比较研究的策略阐述ansacarbamitocins生物合成的后修饰机理和顺序，并利用合成生物学的相关技术，对安丝菌素分子结构进行重新设计，实现与抗体直接耦联的目的，并为药物研发提供资源。我们完成了ansacarbamitocin的生物合成基因簇的克隆和基因功能解析、通过组合生物合成获得ansamitocin的新结构衍生物、氨甲酰基转移酶的异源表达和结晶、催化机制的初步解析、安丝菌素产量提高的途径优化、安丝菌素产生菌菌丝断裂机制及其形态工程、抗肿瘤安莎类格尔登霉素的产量提高。上述研究成果为进一步解析安丝菌素及其类似物的生物合成机制、定向结构和活性优化、高产改造等奠定了良好的基础。.