非核糖体多肽Surugamides生物合成基因簇镶嵌式结构的解析

Elucidation of an intercalation structure of the gene cluster for nonribosomal peptide Surugamides biosynthesis

【目的】多肽化合物Surugamides(sgm)生物合成基因簇包含4个非核糖体多肽合酶(NRPS)基因surA–D,负责2个NRPS生物合成途径。已有报道确认surA基因与SurugamideA产物相关,而surB基因与sgm F产物相关,但对surC和surD基因功能的归属尚没有实验证据。本工作拟在之前研究的基础上进一步确认surA和surD负责Surugamide A产物生物合成,为基因工程改造Surugamides生物合成途径以及研究其NRPS蛋白之间的识别机制提供理论依据。【方法】从海绵中分离放线菌并通过16S rRNA基因序列比对分析其分类单元。通过在线数据库antiSMASH分析基因组序列,发现天然产物生物合成基因簇。通过UPLC-Q-TOF-MS和^(13)CNMR鉴定化合物结构。把构建完成的同源重组双交换质粒导入链霉菌宿主后筛选基因缺失或替换突变株。【结果】从胄甲海绵来源链霉菌S.albidoflavus LHW3101基因组中发现了Surugamides生物合成基因簇,确认了该菌株发酵产物中的化合物sgmA和sgm F。构建了surB和surC基因同时缺失的突变株RJ9,发现RJ9不再产sgm F而仍然产Surugamide A。在缺失突变surB和surC基因的同时在surD基因前引入了组成型强启动子ermEp*,结果发现RJ9产SurugamideA水平是野生型菌株的约2倍。【结论】确认了surB和surC基因与sgmA产物无关。在surD基因前引入强启动子后显著提高了SurugamideA的产量,提示surD基因与sgmA产物相关,结合已报到surA基因与Surugamide A产物相关的证据,进一步确认了surA和surD基因负责Surugamide A生物合成的推论。

[Objective]The Surugamides(sgm)biosynthetic gene cluster has been reported to contain four NRPS genes surA-D which give two unrelated NRPS pathways.It was experimentally confirmed that surA and surB were essential for Surugamide A and Surugamide F production respectively.However,the functions of surC and surD genes are not verified with experimental evidence.This work is designed to confirm if surA and surD genes are responsible for Surugamide A biosynthesis so as to pave the way to either genetically engineering of Surugamides biosynthetic pathways or study the recognition mechanism of their NRPS proteins.[Methods]The Actinomycetes isolated from marine sponge were identified by analyzing their 16 S rRNA gene sequences.The natural product biosynthetic gene clusters were analyzed by submitting genomic sequences to the online database antiSMASH.The chemical structures of isolated compounds were elucidated by UPLC-Q-TOF-MS and 13C NMR.To generate the mutation of gene deletion and replacement,a plasmid was constructed with two fragments used for target homologous recombination.The plasmid was then transformed into the target strain for screening of double-crossover mutants.[Results]We discovered Surugamides gene cluster from the genome of Streptomycesalbidoflavus LHW3101 isolated from marine sponge Dactylospongia elegans.The compound Surugamide A and Surugamide F were then identified from the fermentation extract of the strain.The surB and surC gene in Surugamides gene cluster were replaced with a constitutive and strong promoter ermEp*,which was located justbefore the transcription frame of surD gene.The resulted mutant RJ9 lost the production of Surugamide F but kept on producing Surugamide A with around two-fold yield of the wide type strain.[Conclusion]Gene surB and surC are verified to be not related to sgm A production.It is reported that surA is essential for Surugamide A production.And a remarkable yield improvement for Surugamide A was achieved after introducing a strong promoter before the open reading frame of surD in this work.Therefore we concluded that surA and surD take charge of the biosynthesis of Surugamide A.