紅樹林來源streptomycessp.ouc6819中drimentines類化合物生物合成研究

1、紅樹林來 紅樹林來源Streptomyces Streptomyces Streptomyces Streptomyces sp.OUC681 sp.OUC681 sp.OUC681 sp.OUC6819 9 9 9中Drimentine Drimentine Drimentine Drimentines s s s類化合 類化合物生物合成研究 物生物合成研究摘 要Streptomyces sp. OUC6819 分離自我國南海紅樹林底

2、泥之中，前期研究結(jié)果表明該菌株能夠產(chǎn)生 Drimentines 類化合物。 該類化合物具有 terpenoid-DKP 的雜合結(jié)構(gòu)，并有廣泛而顯著的抗細(xì)菌、抗真菌、抗腫瘤、殺昆蟲和殺寄生蟲等生物活性，Drimentines 的獨特結(jié)構(gòu)與顯著活性引起了科學(xué)家的關(guān)注。本論文以南海紅樹林鏈霉菌 Streptomyces sp. OUC6819 為研究對象， 對其中 Drimentines 類化合物的生物合成進(jìn)行了初步研究，取得了以下研究結(jié)果：

3、1.以 SuperCos1 為載體，構(gòu)建了 Streptomyces sp. OUC6819 的基因組文庫。提取 Streptomyces sp. OUC6819 基因組 DNA，并用 Sau3AⅠ進(jìn)行部分酶切后， 脫磷，回收 30-50kb 基因組片段，與經(jīng) XbaⅠ酶切脫磷后并用 BamHⅠ酶切處理的SuperCos1 相連，經(jīng)噬菌體包裝后侵染 E.coli Trans10，構(gòu)建成基因組文庫，對構(gòu)建成的文庫的質(zhì)量進(jìn)行鑒定，文庫隨機(jī)性

4、良好，效價為 0.6×107cfu/mL，達(dá)到建庫要求。這為基因簇的克隆、基因功能研究與異源表達(dá)奠定了基礎(chǔ)。2.結(jié)合 Drimentines 化學(xué)結(jié)構(gòu)信息及生物信息學(xué)分析，從 Streptomyces sp.OUC6819 基因組掃描序列中定位了可能的 Drimentines 生物合成基因簇，從Streptomyces sp. OUC6819 的基因組文庫中克隆了該基因簇，并進(jìn)行了基因組成與基因功能預(yù)測。推測該基因簇共 33k

5、b，其中包含了 23 個開放閱讀框(OpeningReading Frames,ORFs)：driB,C,D,E,F 基因可能與倍半萜 結(jié)構(gòu)的合成相關(guān)，driK,M,N,O,P,V 基因可能參與二酮哌嗪骨架合成，driJ,L 基因負(fù)責(zé)合成色氨酸，并含有 driA,Q,T,W 四個調(diào)控基因及可能與抗性相關(guān)的 driS。通過菌落 PCR 對基因組文庫進(jìn)行篩選，篩選得到 6 個陽性克隆。3.采用 PCR-targeting 策略， 構(gòu)建了用于

6、基因 driB （編碼聚異戊二烯焦磷酸合酶） 阻斷 雙交 換的 重 組 cosmids，并將 driB 重組 cosmid 轉(zhuǎn)入 Streptomycessp.OUC6819 中，得到了 driB 基因阻斷突變株。采用放線菌Ⅱ號培養(yǎng)基對野生型菌株和△driB 突變株進(jìn)行發(fā)酵，發(fā)酵產(chǎn)物 HPLC-MS 檢測分析表明， △driB 突變S S S Study tudy tudy tudy on on on on the the the th

7、e biosynthesis biosynthesis biosynthesis biosynthesis of of of of Drimentines Drimentines Drimentines Drimentines from from from from mangrove mangrove mangrove mangroveconservation conservation conservation conservation

8、 derived derived derived derived Streptomyces Streptomyces Streptomyces Streptomyces sp.OUC6819 sp.OUC6819 sp.OUC6819 sp.OUC6819Abstract Abstract Abstract AbstractStreptomyces sp.OUC6819 was isolated from the mangrove se

9、diment of SouthChina Sea. It produces terpenoid-diketopiperazine hybrid compounds Drimentines,which exhibit antibacterial, antifungal, anticancer, and anthelmintic activities. Due totheir unique heterozygosity structure

10、and significant bioactivities, Drimentines haveattracted much attention. To reveal the biosynthetic mechanism of Drimentines inStreptomyces sp.OUC6819, we have carried out the following studies:1.The genomic library of S

11、treptomyces sp.OUC6819 was constructed. TotalDNA of Streptomyces sp.OUC6819 was extracted and partially digested by restrictionendonuclease Sau3AI, and then DNA fragments at the size of 30-50kb were recoveredand ligated

12、with SuperCos1, which was digested by XbaI, dephosphorylated andredigested by BamHI. The ligation product was packaged into phage particles withLambda Packaging Extracts, which was then transfected E. coli Trans l0 to yi

13、eld thegenomic library of Streptomyces sp.OUC6819. The titer of the library was 6.0×106cfu/mL, meeting the requirements as a standard genomic library. The clones werestored at -80 ℃. Construction of the genomic libr

14、ary of Streptomyces sp.OUC6819laid the foundation for cloning of biosynthesis gene cluster of secondary metabolitesand their heterologous expression.2. By using strategies of chemical informatics and bioinformatics analy

15、sis, thebiosynthetic gene cluster of Drimentines was identified and isolated from the genomeof Streptomyces sp.OUC6819. The gene cluster is about 33 kb, consisting of 23 openreading frames (ORFs): driB, C, D, E, F are pr

16、oposed to be associated with thebiosynthesis of the sesquiterpene structure; driK, M, N, O, P, V are proposed to beinvolved in the biosynthesis of diketopiperazine skeleton; driJ and driL areresponsible for the synthesis