Biosynthesis of HSAF, an antifungal natural product with a novel mode of action

具有新颖作用方式的抗真菌天然产物 HSAF 的生物合成

• 批准号: 7638002
• 负责人: LIANGCHENG DU
• 金额: $ 7.07万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-17 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7638002
• 关键词: Biosynthesis; HSAF; antifungal; natural; product

DESCRIPTION (provided by applicant): This proposal describes a research plan to biosynthesize HSAF (dihydromaltophilin), a broad spectrum antifungal natural product with a new mode of action. HSAF was isolated from Lysobacter enzymogenes C3, a bacterium used in the biological control of fungal diseases in agriculture. HSAF exhibits potent activities against a wide range of fungal species, including the life-threatening human pathogen, Aspergillus fumigatus, and shows a novel mode of action by disrupting the polarized growth of filamentous fungi. The antifungal activity of HSAF is mediated through a distinct ceramide synthase that is only found in the filamentous fungi. The disruption of this ceramide synthase gene leads to the depletion of a group of sphingolipids that are probably required for polarized growth of fungi. Fungal sphingolipids are structurally distinct from mammalian sphingolipids and represent a new target for antifungal drugs. HSAF has a complex chemical structure, including a tricyclic system fused to a macrolactam containing a tetramic acid, which is different from any existing antifungal drugs. Thus, HSAF has an unprecedented mode of action and a new chemistry, which are criteria for new antifungal agents. However, chemical total synthesis of HSAF is not feasible for commercial production due to its highly complex structure. Biosynthesis via microbial fermentation is a viable approach, which requires an understanding of the biosynthetic mechanism. The long-term goal is to exploit the potential of the tetramic acid-containing macrolactams as a new class of antifungal drugs. The objective of this project is to determine the mechanism for the formation of HSAF tetramic acid and macrolactam and to test the feasibility of HSAF biosynthetic engineering in L. enzymogenes C3. Chemical total synthesis of HSAF is not feasible for commercial production due to its highly complex structure. Biosynthesis via microbial fermentation is a viable approach, which requires an understanding of the biosynthetic mechanism. The formation of tetramic acid and the macrolactam is the key step in HSAF biosynthesis, and the feasibility test for biosynthetic engineering in the host is also essential for the long-term goal. Two specific aims are outlined in the proposal to realize the objective of the project. The Specific Aim 1 is to determine the reactions catalyzed by the nonribosomal peptide synthetase (NRPS) of HSAF synthase. A key feature of HSAF is that it has two amides that are formed from the same amino acid (ornithine). This is distinct from other tetramic acid-containing polyketides. The Specific Aim 2 is to replace the NRPS module to test the feasibility of synthesizing new HSAF analogs in L. enzymogenes. HSAF belongs to a group of natural products with unique structural features and diverse biological activities, such as the anticancer agents discodermide and cylindramide A from marine sponges and the antiprotozoal agent ikarugamycinfrom Streptomyces. None of these products have been studied for their biosynthetic mechanism. The proposed studies will not only determine the mechanism for a key step in HSAF biosynthesis and test the feasibility for producing new HSAF analogs, but also provide information for the biosynthesis of the other complex natural products. PUBLIC HEALTH RELEVANCE The over reliance on a small set of targets in the current therapeutics for fungal infections in humans has led to the alarming increase of drug resistance, especially in AIDS patients and people using invasive medical devices and implants. HSAF provides a new opportunity to combat the drug resistance because it is an antifungal natural product with a new mode of action and a new chemistry. The proposed studies will help develop strategies for the preparation of this product and its analogs to treat fungal infections.

描述（由申请人提供）：本提案描述了生物合成HSAF（二氢麦芽嗜酸粒细胞蛋白）的研究计划，HSAF是一种具有新作用模式的广谱抗真菌天然产物。HSAF分离自产酶溶杆菌C3，一种用于农业真菌病害生物防治的细菌。HSAF对多种真菌物种表现出有效的活性，包括威胁生命的人类病原体烟曲霉，并通过破坏丝状真菌的极化生长显示出一种新的作用模式。HSAF的抗真菌活性通过仅在丝状真菌中发现的独特的神经酰胺合酶介导。这种神经酰胺合酶基因的破坏导致一组鞘脂的消耗，这可能是真菌的极化生长所需的。真菌鞘脂在结构上与哺乳动物鞘脂不同，是抗真菌药物的新靶点。HSAF具有复杂的化学结构，包括与含有特特拉姆酸的大环内酰胺融合的三环系统，这与任何现有的抗真菌药物不同。因此，HSAF具有前所未有的作用模式和新的化学性质，这是新抗真菌剂的标准。然而，HSAF的化学全合成由于其高度复杂的结构而不适于商业化生产。通过微生物发酵进行生物合成是一种可行的方法，这需要了解生物合成机制。长期目标是开发含特特拉姆酸的大环内酰胺作为一类新的抗真菌药物的潜力。本项目的目的是确定HSAF特特拉姆酸和大环内酰胺的形成机理，并检验HSAF生物合成工程在L.酶基因C3。HSAF的化学全合成由于其高度复杂的结构而不适于商业化生产。通过微生物发酵进行生物合成是一种可行的方法，这需要了解生物合成机制。特特拉姆酸和大环内酰胺的形成是HSAF生物合成的关键步骤，在宿主中进行生物合成工程的可行性试验也是实现长期目标的必要条件。为实现该项目的目标，建议中概述了两个具体目标。具体目的1是确定HSAF合成酶的非核糖体肽合成酶（NRPS）催化的反应。HSAF的一个关键特征是它有两个由相同氨基酸（鸟氨酸）形成的酰胺。这与其他含特特拉姆酸的聚酮化合物不同。具体目标2是替换NRPS模块以测试在L中合成新HSAF类似物的可行性。酶基因HSAF是一类具有独特结构特征和多种生物活性的天然产物，如海绵中的抗癌剂discodermide和cylindramide A以及链霉菌中的抗原虫剂ikarugamycin。这些产品中没有一个被研究过它们的生物合成机制。这些研究不仅将确定HSAF生物合成中关键步骤的机制，并测试产生新的HSAF类似物的可行性，而且还为其他复杂天然产物的生物合成提供信息。 公共卫生相关性在目前人类真菌感染的治疗中，过度依赖一小部分靶标，导致耐药性惊人地增加，特别是在艾滋病患者和使用侵入性医疗器械和植入物的人群中。HSAF提供了一个新的机会，以打击耐药性，因为它是一种抗真菌的天然产物，具有新的作用模式和新的化学。拟议的研究将有助于制定制备该产品及其类似物以治疗真菌感染的策略。