Optimization of the Production and Isolation of the Novel Antifungal Occidiofungin

 描述(由申请人提供)：Occidiofunin是一种环状非核糖体合成的抗真菌多肽，具有亚微摩尔杀菌活性，对广泛的真菌具有杀菌活性。食用菌是由革兰氏阴性菌污染伯克霍尔德氏菌产生的。通过我们的结构表征研究，确定了球孢菌素具有独特的化学成分。我们的研究还揭示了球孢菌素具有新的作用机制。奥西芬净是一种对氟康唑和卡泊芬净耐药的白色念珠菌有效的抗真菌药物。食用菌通过诱导酵母细胞的凋亡来触发细胞死亡。超过10毫克/公斤的剂量对小鼠的毒性最小。此外，佐菌素还可以减少感染小鼠肾脏中的光滑念珠菌载量。所有这些研究都表明有必要进一步开发这种新化合物的临床前阶段。这种独特的抗真菌化合物的进一步研究的主要障碍是培养液产率较低。本应用的目的是提高发酵后球孢菌素的产量和纯度。将探索三种方法来提高抗真菌化合物的产量。首先，将对培养基组分进行筛选，以确定它们对抗真菌化合物生产水平的影响。其次，将使用一种基于分子的方法来确定是否有可能通过对污染芽孢杆菌的遗传操作来增加球孢菌素的产量。我们的第三个方法是开发一个异源表达系统。应用的最后一个组成部分是确定从发酵液中提取和纯化抗真菌化合物的最有效方法。这些研究将使球孢菌素的探索性构效关系(SAR)研究成为可能。这项工作对于进一步研究开发抗真菌化合物作为一种新的治疗方法是必要的。

 DESCRIPTION (provided by applicant): Occidiofungin is a cyclic nonribosomally synthesized antifungal peptide with submicromolar fungicidal activity against a broad spectrum of fungi. Occidiofungin is produced by the Gram-negative bacterium Burkholderia contaminans. From our structural characterization studies, occidiofungin was determined to have a unique chemical composition. Our studies have also revealed that occidiofungin has a novel mechanism of action. Occidiofungin is a potent antifungal against fluconazole and caspofungin-resistant Candida albicans. Occidiofungin triggers cell death by inducing apoptosis in yeast cells. Occidiofungin has minimal toxicity in mice at doses exceeding 10 mg/kg. Furthermore, occidiofungin was shown to reduce Candida glabrata load in the kidneys of infected mice. All these studies point to the need to further the preclinical development of this novel compound. The major impediment to furthering investigational studies on this unique antifungal compound is the poor yield from culture liquor. The goal of this application is to enhance the yield and purity of occidiofungin following fermentation. Three approaches will be explored to increase the yield of the antifungal compound. First, media components will be screened to determine their effect on the production levels of the antifungal compound. Second, a molecular based approach will be used to determine whether an increase in production of occidiofungin is possible through genetic manipulation of B. contaminans. Our third approach is to develop a heterologous expression system. The last component of the application is to determine the most effective means of extracting and purifying the antifungal compound from the fermentation liquor. These studies will enable exploratory structure-activity relationship (SAR) studies on occidiofungin. This work is necessary for furthering additional studies aimed at developing the antifungal compound as a new therapeutic.