中國科學(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生研究員領(lǐng)導(dǎo)的團(tuán)隊在次生代謝產(chǎn)物的生物合成研究方面取得重要進(jìn)展,。該團(tuán)隊完成了重要抗菌劑臺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,。這項研究的部分成果已于近日在線發(fā)表于Journal of the American Chemical Society ,。
臺勾霉素（tiacumicins）由放線菌指孢囊菌NRRL 18085產(chǎn)生，具有抗各種革蘭氏陽性菌的活性,，針對艱難梭菌引起的腹瀉的療效優(yōu)于萬古霉素,，目前已經(jīng)在美國進(jìn)入三期臨床試驗。張長生課題組從指孢囊菌NRRL 18085中克隆和鑒定了長約111 kb的DNA片段,，包含了50個開放閱讀框（ORFs）,，涵蓋了完整的臺勾霉素（tiacumicins）生物合成基因簇。通過構(gòu)建指孢囊菌的遺傳操作體系并進(jìn)行基因敲除突變,，研究人員確定了臺勾霉素生物合成基因簇的邊界,，證實了其中31個ORF與臺勾霉素的生物合成相關(guān)，同時從7個關(guān)鍵基因的突變株中分離鑒定了18個臺勾霉素新結(jié)構(gòu)類似物,，從而清晰地闡明了2個糖基轉(zhuǎn)移酶(TiaG1和TiaG2),，2個細(xì)胞色素P450氧化酶（TiaP1和TiaP2），1個?；D(zhuǎn)移酶（TiaS6）,，1個C-甲基轉(zhuǎn)移酶（TiaS2）和1個鹵化酶（TiaM）的體內(nèi)功能。另外，體外生化實驗表明,，鹵化酶TiaM能夠以脫氯的臺勾霉素作為底物進(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)并證實了具有后修飾功能的鹵化酶。這項研究揭示了臺勾霉素的生物合成途徑和生物合成機理,，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,。
這項研究獲得了國家自然科學(xué)基金、中國科學(xué)院“百人計劃”和知識創(chuàng)新工程項目以及國家重點基礎(chǔ)研究發(fā)展計劃（973計劃）等項目的支持,。（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.
