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The ultimate objective of this project is to discover new and structurally diverse hybrid isoprenoid (HI) antibiotics from marine actinomycetes. The research focuses on an unusual group of marine actinomycetes that were discovered in the PIs laboratory and have tentatively been called MAR4. For reasons that are not yet entirely clear, MAR4 strains have proven to be a rich source of HI secondary metabolites, which are generally rare among bacteria. This proposal takes a unique approach that couples creative culturing techniques and poorly studied environmental resources with molecular approaches to rapidly identify strains capable of producing HI secondary metabolites. These strains then become the focus of detailed structure elucidation and biosynthetic studies that will reveal the mechanisms by which the compounds are assembled. The enzymes discovered as part of these studies subsequently have the potential to be used as biochemical tools to create additional structural diversity. The proposed studies target the discovery of a wide range of HI secondary metabolites including compounds with N and C-prenylation, prenylated pyroles, phenazines, and peptides. The results may include the discovery of diverse new chemical structures within the broad HI class. Major objectives include detailed phylogenetic analyses of MAR4 strains, which will expand our understanding of the diversity of bacteria that produce HIs. A related objective is the biochemical characterization of gene clusters associated with HI biosynthesis and the cloning of specificenzymes for use as biochemical tools. The project goals remained the same during this reporting period. These include an assessment of the diversity of MAR4 marine actinomyetes in ocean sediments collected off the coast of California using both culture and culture independent methods. Goals include the collection of new sediment samples and attempts to draw correlations between the abundance of MAR4 strains and environmental parameters. Additional goals include the identification of hybrid isoprenoid (HI) secondary metabolites produced by MAR4 strains and analyses of the relationships between MAR4 phylotype and the suite of secondary metabolites produced. We continue to work on pathways associated with HI biosynthesis, with a focus on strain CNH-189, which produces the unusual merochlorin class of compounds. |
| 這個項(xiàng)目的最終目標(biāo)是從海洋放線菌中發(fā)現(xiàn)新的和結(jié)構(gòu)多樣化的混合類異戊二烯。這項(xiàng)研究的對象是一種特殊的海洋放線菌,它是由PIs實(shí)驗(yàn)室發(fā)現(xiàn)的,暫定取名為MAR4,主要原因是目前還沒有完全弄清楚,MAR4系被證明含有豐富的HI次生代謝產(chǎn)物,這在一般的細(xì)菌中時很罕見的,這個方案的特別之處在于,兼具創(chuàng)造行的培養(yǎng)技術(shù),以及在惡劣的研究環(huán)境下能夠利用分子分析方法迅速的分析出其制造混合類異戊二烯二級產(chǎn)物的能力,這樣就能對MAR4系進(jìn)行具體結(jié)構(gòu)分析和生物合成研究,進(jìn)而通過這些結(jié)果對其進(jìn)行合成。隨后,在這個研究中發(fā)現(xiàn)的酶很有作為生化工具創(chuàng)造其它結(jié)構(gòu)復(fù)雜物質(zhì)的潛力。這個項(xiàng)目的研究目的是發(fā)現(xiàn)一種包含N和c-異戊烯、異戊吡咯、吩嗪 、肽的泛HI二級產(chǎn)物,結(jié)果也許會包含HI家族中的結(jié)構(gòu)多樣化的新物質(zhì)。主要目標(biāo)還包括對MAR4系的具體進(jìn)化分析,這能夠幫助我們了解更多能夠產(chǎn)生HIs的多樣化細(xì)菌,相關(guān)結(jié)果是與HIs合成相關(guān)基因的生化表征和以及克隆出這種能夠作為生化工具的酶,這個項(xiàng)目還在研究階段的是,利用對從加利福尼亞海岸沉積物中收集的MAR4放線菌進(jìn)行培養(yǎng)或者單獨(dú)培養(yǎng)的方式對其多樣性進(jìn)行評估,目標(biāo)包括收集新的泥沙樣本,以試圖找出MAR4系豐富程度與環(huán)境參數(shù)的關(guān)系。另外還包括鑒定MAR4系所產(chǎn)生的HI次生代謝物和分析MAR4放線菌和生產(chǎn)次生代謝產(chǎn)物的工具的關(guān)系。我們將繼續(xù)研究HIs合成的相關(guān)方法,主要集中在對CNH-189系的研究,它能夠產(chǎn)生一種特殊的酶。 |
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