中國(guó)科學(xué)院南海海洋研究所海洋微生物代謝工程與組合生物合成課題組張長(zhǎng)生研究員領(lǐng)導(dǎo)的團(tuán)隊(duì)在次生代謝產(chǎn)物的生物合成研究方面取得重要進(jìn)展,。該團(tuán)隊(duì)完成了重要抗菌劑臺(tái)勾霉素的生物合成基因簇的克隆,、鑒定和關(guān)鍵酶的功能闡述。這項(xiàng)研究的部分成果已于近日在線發(fā)表于Journal of the American Chemical Society ,。
臺(tái)勾霉素（tiacumicins）由放線菌指孢囊菌NRRL 18085產(chǎn)生,，具有抗各種革蘭氏陽(yáng)性菌的活性,，針對(duì)艱難梭菌引起的腹瀉的療效優(yōu)于萬(wàn)古霉素，目前已經(jīng)在美國(guó)進(jìn)入三期臨床試驗(yàn),。張長(zhǎng)生課題組從指孢囊菌NRRL 18085中克隆和鑒定了長(zhǎng)約111 kb的DNA片段,，包含了50個(gè)開(kāi)放閱讀框（ORFs），涵蓋了完整的臺(tái)勾霉素（tiacumicins）生物合成基因簇,。通過(guò)構(gòu)建指孢囊菌的遺傳操作體系并進(jìn)行基因敲除突變,，研究人員確定了臺(tái)勾霉素生物合成基因簇的邊界，證實(shí)了其中31個(gè)ORF與臺(tái)勾霉素的生物合成相關(guān),，同時(shí)從7個(gè)關(guān)鍵基因的突變株中分離鑒定了18個(gè)臺(tái)勾霉素新結(jié)構(gòu)類似物,，從而清晰地闡明了2個(gè)糖基轉(zhuǎn)移酶(TiaG1和TiaG2)，2個(gè)細(xì)胞色素P450氧化酶（TiaP1和TiaP2）,，1個(gè)?；D(zhuǎn)移酶（TiaS6），1個(gè)C-甲基轉(zhuǎn)移酶（TiaS2）和1個(gè)鹵化酶（TiaM）的體內(nèi)功能,。另外,，體外生化實(shí)驗(yàn)表明，鹵化酶TiaM能夠以脫氯的臺(tái)勾霉素作為底物進(jìn)行兩次有序的鹵化反應(yīng),，從而首次在原核生物中發(fā)現(xiàn)并證實(shí)了具有后修飾功能的鹵化酶,。這項(xiàng)研究揭示了臺(tái)勾霉素的生物合成途徑和生物合成機(jī)理，充分展示了組合生物合成技術(shù)在豐富天然產(chǎn)物結(jié)構(gòu)多樣性方面的潛力,。
這項(xiàng)研究獲得了國(guó)家自然科學(xué)基金,、中國(guó)科學(xué)院“百人計(jì)劃”和知識(shí)創(chuàng)新工程項(xiàng)目以及國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃（973計(jì)劃）等項(xiàng)目的支持。（生物谷Bioon.com）
生物谷推薦原文出處：
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.
