本文選題：吩嗪 + 抗生素溶桿菌�。� 參考：《南京農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：溶桿菌屬由Christensen和Cook在1978年建立分類,屬于黃單胞科,γ-變形菌門。溶桿菌在不同的棲息地普遍存在,如海洋、土壤、瀝青坑、污泥堆肥、火山灰以及水生環(huán)境。溶桿菌具有獨(dú)特的特征,如65-72%的高G+C含量、無鞭毛、滑行運(yùn)動(dòng),產(chǎn)生大量胞外水解酶及活性次級(jí)代謝產(chǎn)物。溶桿菌作為潛在的生防菌株和活性天然產(chǎn)物來源日益受到廣泛關(guān)注�？股厝軛U菌OH13分離于水稻根部土壤,對(duì)植物病原細(xì)菌和真菌展現(xiàn)了有效的生防活性。我們利用葡聚糖凝膠層析柱和高效液相色譜從抗生素溶桿菌OH 13中分離純化了六種化合物。通過NMR和MS分析,這些化合物被鑒定為吩嗪類物質(zhì),分別為:(1)1-羥基-6-甲氧基-5,10-二氧吩嗪;(2) 1-羥基-6-甲氧基-10-氧基吩嗪;(3) 1,6-二羥基-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-羥基-6-甲氧基吩嗪�；衔�1-4為氮氧化吩嗪,其中化合物1與已報(bào)道的myxin結(jié)構(gòu)一致,對(duì)多種微生物都具有很強(qiáng)的抗生素活性�；衔�3為已知的抗生素iodinin,對(duì)于革蘭氏陽性細(xì)菌和幾種放線菌及真菌展現(xiàn)很強(qiáng)的抗生素活性。盡管化合物4和5已被化學(xué)合成,但其首次作為天然產(chǎn)物被發(fā)現(xiàn)。吩嗪類天然產(chǎn)物,因?yàn)槠淇辜?xì)菌、抗真菌、抗病毒、抗腫瘤活性已被廣泛研究,特別是來自莽草酸途徑的吩嗪-1-羧酸(PCA)和吩嗪-1,6-二羧酸(PDC)。一個(gè)保守的基因簇,phzABCDEFG,負(fù)責(zé)PCA和PDC的生物合成。PCA和PDC被認(rèn)為是其它更復(fù)雜吩嗪物質(zhì)的合成前體。然而對(duì)于氮氧化吩嗪的生物合成機(jī)制我們是知之甚少的,如myxin (1)和 iodinin (3)。除了氮氧化吩嗪,其它包含芳香族氮氧化基團(tuán)的天然產(chǎn)物是非常稀少的,但通常具有很強(qiáng)的活性。芳香族氮氧化物因?yàn)槠渚哂杏缮镞�原激活的、缺氧選擇性的DNA損傷特性,使得其成為很值得研究的一類抗腫瘤類化合物。然而,對(duì)于這些芳香族氮氧化物的氮氧化機(jī)制仍是不清楚的,因此我們對(duì)抗生素溶桿菌OH13中氮氧化吩嗪類物質(zhì)的氮氧化機(jī)制進(jìn)行研究。通過對(duì)OH13基因組進(jìn)行生物信息學(xué)分析,我們發(fā)現(xiàn)一個(gè)包含10個(gè)基因的基因簇(LaPhz)可能負(fù)責(zé)吩嗪類物質(zhì)的生物合成。這個(gè)LaPhz基因簇包含六個(gè)假定的核心基因,LaPhzB-G,以及四個(gè)假定的修飾基因, LaPhzNO1、LaPhzS、LaPhzX和 LaPhzM。為 了證明 LaPhz 基因簇負(fù)責(zé)OH13中吩嗪物質(zhì)的生物合成,我們首先突變了三個(gè)核心基因,LaPhzC、LaPhzD和LaPhzB。HPLC分析其代謝產(chǎn)物,結(jié)果表明LaPhzC和LaPhzB突變體不能產(chǎn)生吩嗪類物質(zhì),而LaPhzD突變體產(chǎn)生非常少量的化合物2。突變體的活性檢測與HPLC結(jié)果一致,LaaPhzC和LaPhzB突變體喪失了抗細(xì)菌活性,而LaaPhzD突變體僅有微弱的活性。這些結(jié)果證明LaPhz基因簇負(fù)責(zé)抗生素溶桿菌OH13中吩嗪類抗生素的生物合成。LaPhz基因簇中基因LaPhzNO1與編碼環(huán)己酮單加氧酶的基因類似,這類酶是催化Baeyer-Villiger類型反應(yīng)的含黃素蛋白�；騆aPhzNO1突變后,OH13不能產(chǎn)生氮氧化吩嗪,而另外兩個(gè)非氮氧化的吩嗪物質(zhì)產(chǎn)量卻顯著提高。進(jìn)而我們在大腸桿菌中表達(dá)LaPhzNO1,通過體外酶活性檢測,證明LaPhzNO1是依賴于NADPH并且含黃素的氮氧化酶。基因簇中LaPhzS,與另一個(gè)編碼含黃素單加氧酶的PhzS同源,具有脫羧羥基化功能。LaPhzS突變體不能產(chǎn)生化合物1-6,但有PDC產(chǎn)生。這說明PDC是抗生素溶桿菌吩嗪類物質(zhì)的合成前體物質(zhì)。LaPhzS可將PDC轉(zhuǎn)化為1,6-二羥基吩嗪。LaPhzNO1能氧化1,6-二羥基吩嗪為1,6-二羥基-5,10-二氧吩嗪(iodinin,3)。除吩嗪類物質(zhì)外,LaPhzNO1還能催化8-羥基喹啉的氮氧化,這表明LaPhzNO1在化學(xué)酶法合成其它芳香族氮氧化物中具有潛在的應(yīng)用價(jià)值。LaPhzNO1是第一個(gè)經(jīng)過證明的雜環(huán)芳香族天然產(chǎn)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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S476

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