Engineering Biosynthesis of Brefeldin A and Its Analogs

描述（由申请人提供）：聚酮化合物是由聚酮化合物脱氢酶（PKS）产生的一组极其多样化的天然产物。这些化合物是从细菌、放线菌、真菌和植物中分离出来的，作为抗癌、抗病毒、抗细菌、抗真菌、抗寄生虫和免疫抑制剂具有悠久而成功的历史。在过去的十年中，已经从放线菌中分离出许多PKS。它们在组合生物合成中的应用作为天然产物药物发现中潜在的强有力的新工具引起了相当大的兴趣。只有少数PKS系统已被调查丝状真菌，真菌聚酮生物合成的机制仍然知之甚少。布雷菲德菌素A是由布雷菲德正青霉产生的一种大环内酯聚酮化合物，具有广泛的抗菌、抗真菌和抗病毒活性。最近，布雷菲德菌素A可以选择性地诱导某些类型肿瘤细胞凋亡相关的DNA片段化，这一发现极大地激发了人们对布雷菲德菌素A的兴趣，并且布雷菲德菌素A作为抗癌剂正处于临床前开发阶段。不幸的是，布雷菲德菌素A具有一些不期望的药代动力学性质，例如低的水溶性和口服给药后的差的生物利用度。为了解决这些问题，已经采用了复杂的化学方法。另一方面，到目前为止，几乎没有在产生布雷菲德菌素A的E.布雷费尔迪安。 该项目将采用基因克隆、基因破坏、过表达和生物合成的方法来产生适合临床应用的布雷菲德菌素A类似物，并提高布雷菲德菌素A的发酵效价。本课题的具体目标是：1）从大肠杆菌中克隆布雷菲德菌素A PKS;布雷菲德菌素A的方法，2）产生用于在同源和异源宿主中过表达布雷菲德菌素A生产的真菌菌株，和3）通过基因工程修饰产生布雷菲德菌素A的新类似物。

DESCRIPTION (provided by applicant): Polyketides are a group of extremely diverse natural products produced by polyketide synthases (PKSs). These compounds are isolated from bacteria, actinomycetes, fungi, and plants, and have a long and successful history as anticancer, antiviral, antibacterial, antifungal, antiparastic, and immunosuppressive agents. Numerous PKSs have been isolated from actinomycetes in the past decade. Their use in combinatorial biosynthesis has attracted considerable interest as a potential powerful new tool in natural product drug discovery. Only a few PKS systems have been investigated from filamentous fungi, and the mechanisms of fungal polyketide biosynthesis remain poorly understood. Brefeldin A, a macrolide polyketide produced by Eupenicilium brefeldianum, exhibits a wide range of antibiotic, antifungal and antiviral activities. The interest in brefeldin A has been greatly stimulated recently by the finding that it can selectively induce DNA fragmentation associated with apoptosis in certain types of tumor cells, and it is in pre-clinical development as an anticancer agent. Unfortunately, brefeldin A possesses a few undesirable pharmacokinetic properties, such as low aqueous solubility and poor bioavailability after oral administration. To address these issues, complicated chemical methods have been employed. On the other hand, so far almost no physiological and molecular genetic works have been performed in brefeldin A-producing E. brefeldianum. The proposed project will use the combination of gene cloning, gene disruption, over-expression and precursor-directed biosynthesis to generate brefeldin A analogs suitable for clinical applications, and to increase the fermentation titers of brefeldin A. The specific aims of the project are: 1) to clone the brefeldin A PKS from E. brefeldianum, 2) to create fungal strains for the over-expression of brefeldin A production in both homologous and heterologous hosts, and 3) to generate novel analogs of brefeldin A by genetic engineering modification.