基于D-麦角酸肽合成酶的麦角新碱定向生物合成

As a nation essential drug, ergometrine is used for the treatment or prevention of postpartum hemorrhage in clinic due to its pronounced uterotonic effect. The drug is well-known for its lower price and higher effectiveness. After suspension for decades in China, ergometrine production has been resumed by the Sichuan Direction Pharmaceutical Co. Ltd. with the technology transfer from our institute. However, the production of ergometrine in Claviceps purpurea Cp-1 strain bred in our lab is always accompanied by various ergopeptines during the fermentation process. Furthermore, the detailed informations and the types of ergopeptine for which D-lysergyl peptide synthetases (LPS) are responsible in Cp-1 strain still remain unclear. Thus, the encoding sequences of related lps genes (lpsA1 and lpsA2) were acquired through segmented amplification and genome walking method. On the basis of the successful establishment of transformation system of Cp-1 strain, the biological function of lpsA1 and lpsA2 will be illuminated by analyzing the related gene deletion strain, which will provide new theoretical basis of ergopeptines synthesis pathway. Most importantly, based on the knowledge of the biosynthetic mechanism of ergot alkaloids, the production strain will be modified by blocking the related genes of ergopeptine biosynthetic pathway and up-regulating the genes involved in the synthetic pathway of ergometrine. The objective of this project is to obtain the improved strain with the character of increased ergometrine productivity and single ergometrine component through the D-lysergyl peptide synthetase-based directed biosynthesis. The project not only has the important implication for industrial production of ergometrine but also exhibits the referential significance for directed biosynthesis of other clinically important ergot alkaloids.

作为国家基本药物，麦角新碱用于临床预防或治疗产后出血，价格低，药效高，停产数十年后恢复其生产具有重要意义。本项目组筛选到的麦角菌Cp-1菌株已于四川方向药业生产麦角新碱。然而，该菌株在产生麦角新碱时还产生不同种类的麦角肽碱，且其中参与合成麦角肽碱的D-麦角酸肽合成酶（LPS）的具体信息及功能尚不清楚。因此，本项目通过分段扩增并结合染色体步移技术获得该菌株中相关lps（lpsA1和lpsA2）的基因序列，拟在建立遗传转化体系的基础上，通过相关lps基因敲除等研究阐明其功能，为麦角肽碱的生物合成途径解析提供新的理论依据。进一步对Cp-1菌株中麦角碱的生物合成途径进行改造，即敲除相关基因阻断麦角肽碱的合成，过表达麦角新碱合成关键基因，旨在构建产碱成分较为单一且麦角新碱产量提高的优良菌株，实现麦角新碱的定向生物合成。本研究对工业化生产麦角新碱具有重要意义，为临床急需的其他麦角碱的合成提供借鉴。

在特定的液体发酵条件下，黑麦麦角菌Cp-1菌株能产生国家基本药物麦角新碱，还产生不同种类的麦角肽碱。本项目旨在对参与合成麦角肽碱的D-麦角酸肽合成酶（LPS）的功能进行研究，并通过对该菌株进行改造，构建更优良的生产菌株。目前，已取得如下结果：.1. 通过分段扩增并结合染色体步移技术获得Cp-1菌株中相关lps（lpsA1和lpsA2）的基因全序列，结合生物学信息分析的结果表明，同种的不同黑麦麦角菌菌株中，lpsAs的氨基酸序列一致性为78.94%-90.24%，存在序列异质性。.2. 基于同源重组对目的基因进行敲除，经过多次原生质体制备和上千株转化子筛选，得到ΔlpsA1hetero-Cp-1异核体和ΔlpsA2homo-Cp-1同核体。对各菌株发酵产物进行HPLC和LC-MS分析的结果表明，与Cp-1野生型菌株相比，ΔlpsA2homo-Cp-1同核体和ΔlpsA1hetero-Cp-1异核体发酵产物中，麦角肽碱（α-麦角隐亭、β-麦角隐亭和麦角考宁）的产量均有所下降，但均未完全消失，说明LPSA1和LPSA2均参与上述麦角肽碱的合成。ΔlpsA1hetero-Cp-1异核体中各麦角肽碱的产量下降更为明显，且RT-PCR结果表明在整个发酵阶段，lpsA1的基因表达量始终高于lpsA2，暗示着LPSA1在麦角肽碱合成过程中更为重要。同时，敲除菌中麦角新碱的产量均有所增加，说明麦角肽碱合成能力的减弱引起代谢流向麦角新碱的合成方向。鉴于上述三种麦角肽碱结构类似，其区别仅在于链环肽部分的第二个氨基酸的不同，且这些氨基酸结构相似，推测LPSA1-Cp-1和LPSA2-Cp-1具有一定的氨基酸底物结合宽泛性，使其可参与合成多种麦角肽碱。.3. 进一步构建dmaW、lpsB、lpsC过表达质粒，并成功转化入Cp-1菌株中，使这些基因在强启动子Pgpd的作用下完成多拷贝的基因的表达。结果表明过表达lpsB能使麦角新碱的产量提高至原来的2倍。.综上，本项目初步阐明lpsA1和lpsA2功能，通过对该菌株麦角碱的生物合成途径进行改造，获得了产碱成分较为单一且麦角新碱产量提高的菌株，这对工业化生产麦角新碱具有较为重要意义。