Engineering Biosynthesis of Brefeldin A and Its Analogs

说明书(申请人提供)：聚酮是由聚酮合成酶(PKS)产生的一组极其多样的天然产物。这些化合物是从细菌、放线菌、真菌和植物中分离出来的，在抗癌、抗病毒、抗细菌、抗真菌、抗寄生虫和免疫抑制方面有着悠久而成功的历史。在过去的十年里，从放线菌中分离出了大量的PKS。它们在组合生物合成中的应用已经引起了人们的极大兴趣，因为它们是天然产物药物发现中潜在的强有力的新工具。目前从丝状真菌中发现的PKS系统还很少，对真菌聚酮生物合成的机制还知之甚少。Brefeldin A是由Eupenicilium breFeldianum产生的一种大环内酯类聚酮类化合物，具有广泛的抗菌、抗真菌和抗病毒活性。最近，人们对灯盏花素A的兴趣大大增加，因为它可以选择性地诱导某些类型的肿瘤细胞发生与凋亡相关的DNA片段化，并且它正处于临床前开发阶段。不幸的是，灯盏花素A具有一些不良的药代动力学特性，如低水溶解度和口服后生物利用度差。为了解决这些问题，人们采用了复杂的化学方法。另一方面，到目前为止，几乎还没有对产生布雷菲尔丁A的E.breFeldianum进行生理学和分子遗传学研究。该项目将结合基因克隆、基因中断、超表达和前体导向的生物合成来产生适合临床应用的布雷菲尔丁A类似物，并提高布雷菲尔丁A的发酵效价。该项目的具体目标是：1)从短梗霉中克隆布雷菲尔丁A PKS，2)创造在同源和异源宿主中高效表达布雷菲尔丁A的真菌菌株，3)通过基因工程改造产生新的布雷菲尔丁A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.