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

描述（由申请人提供）：本申请描述了一项生物合成HSAF（二氢麦芽酚素）的研究计划，HSAF是一种广谱抗真菌天然产物，具有新的作用方式。HSAF从酶原溶杆菌C3中分离得到，该细菌用于农业真菌病的生物防治。HSAF对多种真菌物种，包括危及生命的人类病原体烟曲霉具有强大的活性，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的抗真菌活性是通过一种独特的神经酰胺合成酶介导的，这种神经酰胺合成酶仅在丝状真菌中发现。这种神经酰胺合成酶基因的破坏导致一组鞘脂的消耗，这可能是真菌极化生长所必需的。真菌鞘脂在结构上不同于哺乳动物鞘脂，是抗真菌药物的新靶点。HSAF具有复杂的化学结构，包括与含有四乙酸的大内酰胺融合的三环体系，这与现有的任何抗真菌药物都不同。因此，HSAF具有前所未有的作用方式和新的化学性质，这是开发新型抗真菌药物的标准。然而，由于HSAF的结构高度复杂，化学全合成尚不能实现商业化生产。微生物发酵生物合成是一种可行的方法，但这需要对生物合成机制的理解。长期目标是开发含四羧酸的大内酰胺类抗真菌药物的潜力。本课题的目的是确定HSAF四酸和大内酰胺的形成机制，并检验在L. enzymatic genes C3中进行HSAF生物合成工程的可行性。由于其结构高度复杂，化学全合成的HSAF难以实现商业化生产。微生物发酵生物合成是一种可行的方法，但这需要对生物合成机制的理解。四羧酸和内酰胺的形成是HSAF生物合成的关键步骤，在宿主体内进行生物合成工程的可行性测试也是实现这一长期目标的必要条件。提案中概述了实现项目目标的两个具体目标。特异性目的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种酰胺，这两种酰胺是由相同的氨基酸（鸟氨酸）形成的。这与其他含四羧酸的聚酮不同。具体目的2是替换NRPS模块，以验证在L. enzymatic genes中合成新的HSAF类似物的可行性。HSAF是一类具有独特结构特征和多种生物活性的天然产物，如来自海绵的抗癌剂discoderamide和圆柱体酰胺a，以及来自链霉菌的抗原虫剂ikarugamycin。这些产物的生物合成机理尚未被研究过。本研究不仅将确定HSAF生物合成关键步骤的机制，验证制备新的HSAF类似物的可行性，而且还将为其他复杂天然产物的生物合成提供信息。

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.