新型抗真菌化合物HSAF的生物合成机制研究

The discovery of new anti-infective drugs is a pressing and continual need for human health due to the constant emergence of drug resistant pathogens. Previously, we isolated HSAF from Lysobacter enzymogenes C3, and identified the 19-gene HSAF cluster. HSAF is a tetramic acid-containing macrolactam fused to a tricyclic system, and belongs to polycyclic tetramate macrolactams (PTM). HSAF exhibits potent inhibitory activities against a wide range of fungi, and shows a novel mode of action. The most striking feature of HSAF gene cluster is the presence of only a single-module polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS), although the biosynthesis of HSAF apparently requires two separate polyketide chains..Therefore, in this project diverse approaches will be taken to define the minimal gene cluster required for HSAF biosynthesis, to interpret the formation mechanism of tetramic acid unit and 5,5,6-tricyclic system, and finally to decipher the biosynthetic mechanism of HSAF. The approaches includ in-frame deletion of the individual genes, heterologous expression of a series of HSAF gene cluster constructs, heterologous expression of key proteins, characterization of the reactions catalyzed by key proteins and so on. The completion of the experiments will presents the first example where a single iterative PKS-NRPS is modulated by a cascade of redox enzymes to assemble two separate polyketide chains that are linked together via two amide bonds on the same amino acid, and establish a new mechanism for the biosynthesis of bacterial hybrid polyketide-peptide. The knowledge obtained here will also set the foundation for more insightful molecular mechanistic studies and biosynthetic engineering of other bioactive PTMs. Meanwhile, the mechanistic understanding should allow transfer of such logic to the discovery of new bioactive NPs and allow optimization of the structure and activity of other anti-infectives.

由于耐药菌的不断出现，新结构和新作用机制抗生素的发现在今天显得尤为迫切。前期，我们从产酶溶杆菌C3中分离得到了化学结构新颖、作用机制独特的强效广谱抗真菌化合物HSAF，并获得了其19个基因的生物合成基因簇。目前，HSAF所属的PTM类抗生素的生物合成研究刚刚起步。本项目将通过基因敲除、基因簇异源表达和关键蛋白的体内外功能研究，明确HSAF最小基因簇组成，揭示tetramic acid单元和多环结构的形成机制，进而阐明HSAF的生物合成机制。本项目的完成将首次证实细菌中存在"重复使用的单模块PKS"-NRPS，同时将确立细菌中单个PKS-NRPS通过与一系列氧化还原酶的相互作用即可合成结构复杂且高度修饰的PTMs的生物合成新模式。同时，也为PTMs的遗传改造和新活性PTMs的获得奠定了理论基础；此外，对于新活性天然产物的发掘和已有次生代谢产物的改良也有着重要的理论和应用价值。

由于耐药菌的不断出现，新结构和新作用机制抗生素的发现在今天显得尤为迫切。前期，我们从产酶溶杆菌C3中分离得到了化学结构新颖、作用机制独特的强效广谱抗真菌化合物HSAF(heat stable antifungal factor)，并定位了其生物合成基因簇，但是HSAF的生物合成机制尚未完全解析。本项目通过对HSAF基因簇在链霉菌中进行异源表达，分离鉴定了HSAF类化合物，检测到了含有tetramic acid结构单元的核心骨架。上述研究结果一方面，明确了HSAF最小基因簇组成；另一方面，证实了“单模块PKS”可以通过重复使用催化合成2条独立的聚酮链，并在NRPS的催化下与1分子鸟氨酸缩合形成含有tetramic acid结构单元的核心骨架，从而揭示了polycyclic tetramate macrolactam (PTM) 类化合物中tetramic acid单元的形成机制。本项目的完成证实了细菌中存在“重复使用的单模块PKS”-NRPS，为挖掘其它未知的PTM类化合物奠定了理论基础。此外，对于新活性天然产物的发掘和已有次生代谢产物的改良也有着重要的理论和应用价值。