假单胞菌新型铁载体7-羟基环庚三烯酚酮的合成调控机理研究

A tremendous variety of Pseudomonads widely distributed in diverse niches, which are closely related to health and medical, biocontrol, and environmental protection, have obtained much attention. Because of the diversity of types and functions of siderophores produced by Pseudomonads, Pseudomonas plays an important role in the researches of bacterial siderophores. In our previous investigations, we had isolated the high-siderophore-yielding Pseudomonas donghuensis strain HYS from environmental water samples, and found that the high-yield non-fluorescent siderophore is 7-hydroxytropolone. The finding that 7-hydroxytropolone could be utilized as a siderophore in Pseudomonads has not been reported, suggesting that 7-hydroxytropolone is a novel siderophore. The preliminary studies indicate that the biosynthesis of 7-hydroxytropolone is related with two gene clusters in the HYS genome. In this project, our efforts will concentrate on the biosynthesis pathway and regulatory mechanism of the high-yield non-fluorescent siderophore 7-hydroxytropolone in strain HYS. The intention and significance of this project are: (1) determining the basic biosynthesis pathway of the novel siderophore in Pseudomonads, and elucidating the key steps in the biosynthesis of bacterial troponoids; (2) elucidating the regulatory mechanism of iron metabolisms with different siderophores in HYS, and providing a guideline for further understandings of environmental adaptation mechanisms of Pseudomonads, and the biosynthesis and application of troponoids.

假单胞菌属细菌分布广、种类多，与医疗健康、生物防治、环境保护等密切相关，一直深受关注。假单胞菌所产铁载体种类和功能多样，相关研究在细菌铁载体研究中占主导地位。本课题组从环境水样中，分离得到高产铁载体东湖假单胞菌HYS菌株，发现高产的非荧光铁载体是7-羟基环庚三烯酚酮，其在假单胞菌中作为铁载体发挥功能属新的发现，是假单胞菌中的新型铁载体。初步研究显示，HYS菌株7-羟基环庚三烯酚酮的合成与基因组上两个基因簇有关。本项目拟在现有工作基础上，研究HYS菌株高产非荧光铁载体7-羟基环庚三烯酚酮的生物合成途径及其调控机理，目的和意义在于：（1）明确假单胞菌新型铁载体生物合成基本途径，解析细菌环庚三烯酮类化合物生物合成过程中的关键步骤；（2）阐明HYS菌株中不同类型铁载体参与铁代谢的调控规律，为深入了解假单胞菌适应环境的多样性机制及具有重要生物学活性的环庚三烯酮类化合物生物合成与应用提供参考。

假单胞菌属细菌广泛分布于自然界中，是一类常见的环境微生物，与人类的生产生活密切相关。该属中一些致病菌在细菌致病机理及生物防治等方面均有重要的理论研究和应用价值。产铁载体是假单胞菌应对复杂多变环境和参与竞争的重要手段，与其致病性密切相关。东湖假单胞菌HYS菌株是本实验室分离鉴定的一株高产铁载体假单胞菌。本项目通过确定HYS菌株中与7-羟基环庚三烯酚酮合成相关的基因簇各基因功能，初步解析了HYS菌株高产非荧光铁载体7-羟基环庚三烯酚酮的生物合成途径及其基本调控机制。明确东湖假单胞菌HYS菌株中7-羟基环庚三烯酚酮基本碳骨架来源于苯乙酸代谢途径，合成的后半程为由含特殊操纵子orf6-9的基因簇1参与的催化反应完成开环、缩合和脱氢过程；揭示了HYS菌株通过不同同工酶和利用两种不同调控家族蛋白LysR和TetR/AcrR相互作用共同调控7-羟基环庚三烯酚酮合成，从而实现对细菌本身具有明显抑制作用的生物活性物质合成的精细化调控方式；利用秀丽隐杆线虫慢致死模型的毒性研究发现，HYS菌株毒性与其产铁载体能力相关；发现7-羟基环庚三烯酚酮合成中最关键的操纵子orf6-9在假单胞菌中极为少见，有可能通过较为特殊的方式演化而来。本项目在7-羟基环庚三烯酚酮生物合成途径和调控机制方面取得的阶段性研究结果为深入了解重要生物学活性次级代谢产物合成调控及假单胞菌适应环境的多样性机制提供了新线索。