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JACS:南海海洋所微生物次生代谢生物合成研究进展

2010/12/30 来源:南海海洋研究所
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中国科学院南海海洋研究所海洋微生物代谢工程与组合生物合成课题组张长生研究员领导的团队在次生代谢产物的生物合成研究方面取得重要进展。该团队完成了重要抗菌剂台勾霉素的生物合成基因簇的克隆、鉴定和关键酶的功能阐述。这项研究的部分成果已于近日在线发表于Journal of the Ame

中国科学院南海海洋研究所海洋微生物代谢工程与组合生物合成课题组张长生研究员领导的团队在次生代谢产物的生物合成研究方面取得重要进展。该团队完成了重要抗菌剂台勾霉素的生物合成基因簇的克隆、鉴定和关键酶的功能阐述。这项研究的部分成果已于近日在线发表于Journal of the American Chemical Society 。

台勾霉素(tiacumicins)由放线菌指孢囊菌NRRL 18085产生,具有抗各种革兰氏阳性菌的活性,针对艰难梭菌引起的腹泻的疗效优于万古霉素,目前已经在美国进入三期临床试验。张长生课题组从指孢囊菌NRRL 18085中克隆和鉴定了长约111 kb的DNA片段,包含了50个开放阅读框(ORFs),涵盖了完整的台勾霉素(tiacumicins)生物合成基因簇。通过构建指孢囊菌的遗传操作体系并进行基因敲除突变,研究人员确定了台勾霉素生物合成基因簇的边界,证实了其中31个ORF与台勾霉素的生物合成相关,同时从7个关键基因的突变株中分离鉴定了18个台勾霉素新结构类似物,从而清晰地阐明了2个糖基转移酶(TiaG1和TiaG2),2个细胞色素P450氧化酶(TiaP1和TiaP2),1个酰基转移酶(TiaS6),1个C-甲基转移酶(TiaS2)和1个卤化酶(TiaM)的体内功能。另外,体外生化实验表明,卤化酶TiaM能够以脱氯的台勾霉素作为底物进行两次有序的卤化反应,从而首次在原核生物中发现并证实了具有后修饰功能的卤化酶。这项研究揭示了台勾霉素的生物合成途径和生物合成机理,充分展示了组合生物合成技术在丰富天然产物结构多样性方面的潜力。

这项研究获得了国家自然科学基金、中国科学院“百人计划”和知识创新工程项目以及国家重点基础研究发展计划(973计划)等项目的支持。

 

原文出处:

J. Am. Chem. Soc. DOI: 10.1021/ja109445q

Characterization of Tiacumicin B Biosynthetic Gene Cluster Affording Diversified Tiacumicin Analogues and Revealing a Tailoring Dihalogenase

Yi Xiao, Sumei Li, Siwen Niu, Liang Ma, Guangtao Zhang, Haibo Zhang, Gaiyun Zhang, Jianhua Ju, and Changsheng Zhang*

CAS Key Laboratory of Marine Bio-resources Sustainable Utilization, RNAM Center for Marine Microbiology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, P.R. China

Abstract

The RNA polymerase inhibitor tiacumicin B is currently undergoing phase III clinical trial for treatment of Clostridium difficile associated diarrhea with great promise. To understand the biosynthetic logic and to lay a foundation for generating structural analogues via pathway engineering, the tiacumicin B biosynthetic gene cluster was identified and characterized from the producer Dactylosporangium aurantiacum subsp. hamdenensis NRRL 18085. Sequence analysis of a 110633 bp DNA region revealed the presence of 50 open reading frames (orfs). Functional investigations of 11 orfs by in vivo inactivation experiments, preliminarily outlined the boundaries of the tia-gene cluster and suggested that 31 orfs were putatively involved in tiacumicin B biosynthesis. Functions of a halogenase (TiaM), two glycosyltransferases (TiaG1 and TiaG2), a sugar C-methyltransferase (TiaS2), an acyltransferase (TiaS6), and two cytochrome P450s (TiaP1 and TiaP2) were elucidated by isolation and structural characterization of the metabolites from the corresponding gene-inactivation mutants. Accumulation of 18 tiacumicin B analogues from 7 mutants not only provided experimental evidence to confirm the proposed functions of individual biosynthetic enzymes, but also set an example of accessing microbial natural product diversity via genetic approach. More importantly, biochemical characterization of the FAD-dependent halogenase TiaM reveals a sequentially acting dihalogenation step tailoring tiacumicin B biosynthesis.

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