抗生素溶杆菌中吩嗪类物质的鉴定及其生物合成机制研究

发布时间：2018-06-25 03:12

本文选题：吩嗪 + 抗生素溶杆菌　；参考：《南京农业大学》2016年博士论文

【摘要】：溶杆菌属由Christensen和Cook在1978年建立分类,属于黄单胞科,γ-变形菌门。溶杆菌在不同的栖息地普遍存在,如海洋、土壤、沥青坑、污泥堆肥、火山灰以及水生环境。溶杆菌具有独特的特征,如65-72%的高G+C含量、无鞭毛、滑行运动,产生大量胞外水解酶及活性次级代谢产物。溶杆菌作为潜在的生防菌株和活性天然产物来源日益受到广泛关注。抗生素溶杆菌OH13分离于水稻根部土壤,对植物病原细菌和真菌展现了有效的生防活性。我们利用葡聚糖凝胶层析柱和高效液相色谱从抗生素溶杆菌OH 13中分离纯化了六种化合物。通过NMR和MS分析,这些化合物被鉴定为吩嗪类物质,分别为:(1)1-羟基-6-甲氧基-5,10-二氧吩嗪;(2) 1-羟基-6-甲氧基-10-氧基吩嗪;(3) 1,6-二羟基-5,10-二氧吩嗪;(4) 1,6-二甲氧基-10-氧基吩嗪;(5) 1,6-二甲氧基吩嗪;(6) 1-羟基-6-甲氧基吩嗪。化合物1-4为氮氧化吩嗪,其中化合物1与已报道的myxin结构一致,对多种微生物都具有很强的抗生素活性。化合物3为已知的抗生素iodinin,对于革兰氏阳性细菌和几种放线菌及真菌展现很强的抗生素活性。尽管化合物4和5已被化学合成,但其首次作为天然产物被发现。吩嗪类天然产物,因为其抗细菌、抗真菌、抗病毒、抗肿瘤活性已被广泛研究,特别是来自莽草酸途径的吩嗪-1-羧酸(PCA)和吩嗪-1,6-二羧酸(PDC)。一个保守的基因簇,phzABCDEFG,负责PCA和PDC的生物合成。PCA和PDC被认为是其它更复杂吩嗪物质的合成前体。然而对于氮氧化吩嗪的生物合成机制我们是知之甚少的,如myxin (1)和 iodinin (3)。除了氮氧化吩嗪,其它包含芳香族氮氧化基团的天然产物是非常稀少的,但通常具有很强的活性。芳香族氮氧化物因为其具有由生物还原激活的、缺氧选择性的DNA损伤特性,使得其成为很值得研究的一类抗肿瘤类化合物。然而,对于这些芳香族氮氧化物的氮氧化机制仍是不清楚的,因此我们对抗生素溶杆菌OH13中氮氧化吩嗪类物质的氮氧化机制进行研究。通过对OH13基因组进行生物信息学分析,我们发现一个包含10个基因的基因簇(LaPhz)可能负责吩嗪类物质的生物合成。这个LaPhz基因簇包含六个假定的核心基因,LaPhzB-G,以及四个假定的修饰基因, LaPhzNO1、LaPhzS、LaPhzX和 LaPhzM。为了证明 LaPhz 基因簇负责OH13中吩嗪物质的生物合成,我们首先突变了三个核心基因,LaPhzC、LaPhzD和LaPhzB。HPLC分析其代谢产物,结果表明LaPhzC和LaPhzB突变体不能产生吩嗪类物质,而LaPhzD突变体产生非常少量的化合物2。突变体的活性检测与HPLC结果一致,LaaPhzC和LaPhzB突变体丧失了抗细菌活性,而LaaPhzD突变体仅有微弱的活性。这些结果证明LaPhz基因簇负责抗生素溶杆菌OH13中吩嗪类抗生素的生物合成。LaPhz基因簇中基因LaPhzNO1与编码环己酮单加氧酶的基因类似,这类酶是催化Baeyer-Villiger类型反应的含黄素蛋白。基因LaPhzNO1突变后,OH13不能产生氮氧化吩嗪,而另外两个非氮氧化的吩嗪物质产量却显著提高。进而我们在大肠杆菌中表达LaPhzNO1,通过体外酶活性检测,证明LaPhzNO1是依赖于NADPH并且含黄素的氮氧化酶。基因簇中LaPhzS,与另一个编码含黄素单加氧酶的PhzS同源,具有脱羧羟基化功能。LaPhzS突变体不能产生化合物1-6,但有PDC产生。这说明PDC是抗生素溶杆菌吩嗪类物质的合成前体物质。LaPhzS可将PDC转化为1,6-二羟基吩嗪。LaPhzNO1能氧化1,6-二羟基吩嗪为1,6-二羟基-5,10-二氧吩嗪(iodinin,3)。除吩嗪类物质外,LaPhzNO1还能催化8-羟基喹啉的氮氧化,这表明LaPhzNO1在化学酶法合成其它芳香族氮氧化物中具有潜在的应用价值。LaPhzNO1是第一个经过证明的杂环芳香族天然产物生物合成中的氮加氧酶。
[Abstract]:The genus of Christensen and Cook was established in 1978, belonging to the family of Xanthomonas and gamma deformable bacteria. The bacteria are ubiquitous in different habitats, such as oceans, soils, bitumen pits, sludge compost, volcanic ash, and aquatic environment. The bacteria have unique characteristics, such as the high G+C content of 65-72%, no flagellum, gliding movement, producing a large number of extracellular As a potential biocontrol strain and the source of active natural products, Bacillus lysin OH13 was isolated from the root soil of rice and showed effective biocontrol activity to plant pathogenic bacteria and fungi. We used glucan gel column and high performance liquid chromatography to resist it. Six compounds were isolated and purified from OH 13. By NMR and MS, these compounds were identified as phenazine compounds: (1) 1- hydroxyl -6- methoxy -5,10- two oxyphenazine; (2) 1- hydroxyl -6- methoxy phenoxazine; (3) 1,6- two hydroxyl -5,10- two oxyphenazine; (4) 1,6- two methoxy phenoxazine; (5) two a Oxygen based phenazine; (6) 1- hydroxyl -6- methoxazine. Compound 1-4 is nitrogen oxide phenazine, in which compound 1 is consistent with reported myxin structure and has strong antibiotic activity to a variety of microbes. Compound 3 is known as the antibiotic iodinin, which shows strong antibiotic activity for Gram-positive bacteria and several actinomycetes and fungi Although compounds 4 and 5 have been chemically synthesized, they have been discovered as natural products for the first time. Phenazine natural products have been widely studied because of their antibacterial, antifungal, antiviral, antitumor activity, especially from the phenazine -1- carboxylic acid (PCA) and phenazine -1,6- two carboxylic acid (PDC) from the shikimic acid pathway. A conservative gene cluster, phzABCDEFG, is responsible for it. The biosynthesis of PCA and PDC biosynthesis of.PCA and PDC is considered as a precursor of other more complex phenazine compounds. However, we know little about the biosynthesis mechanism of azooxazine, such as myxin (1) and iodinin (3). Aromatic nitrogen oxides have become a class of antitumor compounds that are well worth studying because they are activated by biological reduction and the selective DNA damage of hypoxia. However, the mechanism of nitrogen oxidation of these aromatic nitrogen oxides is still unclear. Therefore, we resist the oxidation of nitrogen in OH13. The nitrogen oxidation mechanism of phenazine is studied. By bioinformatics analysis of the OH13 genome, we found that a gene cluster containing 10 genes (LaPhz) may be responsible for the biosynthesis of phenazine. This LaPhz gene cluster contains six hypothetical core genes, LaPhzB-G, and four hypothetical modifier genes, LaPhzNO1 In order to prove that the LaPhz gene cluster is responsible for the biosynthesis of phenazine substances in OH13, LaPhzS, LaPhzX and LaPhzM., we first mutated three core genes, LaPhzC, LaPhzD and LaPhzB.HPLC to analyze their metabolites. The results showed that the mutant of LaPhzC and LaPhzB did not produce phenazine, while the LaPhzD mutant produced a very small amount of compound 2.. The activity detection of the mutant was consistent with the HPLC results, and the LaaPhzC and LaPhzB mutants lost their antibacterial activity, while the LaaPhzD mutant had only a weak activity. These results showed that the LaPhz gene cluster was responsible for the gene LaPhzNO1 and the encoding of cyclohexanone monooxygenase in the biosynthesis.LaPhz gene cluster of phenazine antibiotics in the antibiotic insoluble OH13. Similarly, these enzymes are the flavin proteins that catalyze the Baeyer-Villiger type reaction. After the mutation of the gene LaPhzNO1, OH13 does not produce nitrogen oxide phenazine, and the other two phenazine compounds with non nitrogen oxidation have increased significantly. Then we express LaPhzNO1 in Escherichia coli, and the enzyme activity detection in vitro shows that LaPhzNO1 is dependent on NADPH. And the nitrogen oxidase containing flavin. LaPhzS in the gene cluster is homologous with another PhzS encoding the flavin monooxygenase, and the decarboxylation hydroxylation function.LaPhzS mutant does not produce compound 1-6, but has PDC production. This indicates that PDC is a synthetic precursor of the phenazine substance of the antibiotic,.LaPhzS can convert PDC into 1,6- two hydroxy phenograph. Methazine.LaPhzNO1 can oxidize 1,6- two hydroxyl phenazine to 1,6- two hydroxyl -5,10- two oxophenazine (iodinin, 3). Besides phenazine, LaPhzNO1 can also catalyze the nitrogen oxidation of 8- hydroxyquinoline, which indicates that LaPhzNO1 has potential application value in chemical enzymatic synthesis of other aromatic NOx. It is the first certified heterocyclic aroma. Nitrogen oxygenase in the biosynthesis of natural products of the family.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S476

【参考文献】