恶臭假单胞菌YL19产2种环脂肽的结构鉴定及合成机制研究

The novel structure, action mechanisms, and board activities of cyclic lipopeptides (CLPs) make them the new targets for drug screening. However, the unclear biosynthesis pathways of CLPs limited the screening and modification of high-yielding CLPs producing strains. Thus, it is important to study the biosynthesis mechanism of CLPs. In previous studies, one bacterial strain YL19 had been isolated as it showed the inhibition activates against several kinds of phytopathogenic fungi, several pathogenic bacteria, and even several tumor cell lines. Then, it was identified that YL19 was a novel strain of Pseudomonas putida. Also, CLPs had been identified in the fermentation broth as the active compound produced by YL19. Interestingly, it was rare in Pseudomonas as it was found that YL19 might produce 2 kinds of CLPs. In this project, we plan to isolate and purify the metabolites of YL19, identify the structure of 2 CLPs by mass spectrum, NMR spectrum, and other techniques. Also, the bio-activities of 2 CLPs should be detected and determined. Furthermore, the structures of the CLPs NRPS biosynthesis gene clusters will be identified which might help to reveal the biosynthesis mechanism of one Pseudomonas producing 2 CLPs. This study help to provide the candidates for development of new drugs, to provide new ideas and methods to explore novel CLPs producing strains, to provide theoretical foundation for modification CLPs gene engineering strains.

环脂肽全新的结构类型、作用机制，及广泛的生物活性，使其成为当今新型药物筛选热点。但环脂肽生物合成途径不完全明确，限制了高产菌株的筛选和改造，因此环脂肽生物合成机制是这一领域的重点问题。本课题组筛选到一株细菌YL19，具有抗多种植物病原真菌、细菌，及抑制肿瘤细胞的活性，系统鉴定为恶臭假单胞菌（Pseudomonas putida）新菌株。初步研究表明该菌株胞外生物活性物质主要为环脂肽类化合物，值得关注的是该菌株可能产生2种类型环脂肽，这在假单胞菌中是很少见的。本课题拟对YL19代谢产物进行分离提纯，通过质谱、核磁等波谱技术鉴定2种环脂肽结构；通过抗菌实验等明确2种环脂肽的生物学活性；通过全基因组测序解析YL19环脂肽生物合成基因簇结构，揭示1株假单胞菌产2种环脂肽的合成机制。本研究为环脂肽类药物的研发提供备选化合物、为挖掘产环脂肽的新菌种资源提供新思路和新方法、为功能菌株改造奠定理论基础。

假单胞菌产生的环脂肽有多样的结构类型、广泛的生物活性，是新型药物筛选研发热点。本研究筛选到一株具有较好抗菌、及抑制肿瘤细胞活性的假单胞菌YL19，实验证实该菌株能合成多种环脂肽。本项目对YL19菌株进行了多项分类学鉴定；对YL19的次级代谢产物进行分离纯化，并通过LC-MS实验鉴定代谢物结构；同时通过基因组测序解析YL19基因组信息。结果表明，YL19与Pseudomonas extremaustralis BS2774的OrthoANI值为98%、DDH值为83.45，说明YL19属于P. extremorientalis，但其某些生理代谢指标、及细胞膜脂肪酸等指标与模式菌株存在一定差异。YL19产生10种脂肽，包括3种直链脂肽和7种环脂肽，是viscosin家族的一系列同系物，其中8种为首次报道；通过基因组数据的antiSMASH分析，定位了参与viscosin脂肽合成的基因簇，确定该合成簇是由PKS和NRPS共同组成的，并推导了YL19中viscosin的生物合成途径，与以往报道的P. fluorescens SWB25菌株中的viscosin生物合成途径一致。同时根据基因组数据进行信息学分析，发现YL19中存在转录后水平调控的Gac/Rsm系统，包括GacS/GacA二元调控因子、非编码sRNA、RsmA/RsmE翻译阻遏蛋白等，推测其viscosin合成可能受到Gac/Rsm系统调控；同时还发现YL19中存在一个保守度较高的多功能转录后水平调控因子Hfq、以及热休克蛋白、丝氨酸蛋白酶、磷酸甘油酸脱氢酶、鸟苷四磷酸合成酶/脱氢酶等转录水平调控元件，因此YL19中环脂肽生物合成应该存在多水平、多通路的调控网络。本项目明确了YL19菌株合成环脂肽的种类及结构，为开发具有较好生物活性的小分子环脂肽类化合物提供了物质基础；同时确定了YL19基因组上viscosin合成簇定位及结构、推导了viscosin合成途径，并分析了viscosin合成的调控网络，为功能菌株改造提供了理论基础和实验菌株准备。