海鞘来源放线菌Streptomyces pratensis SCSIO LCY05中skyllamycins的发现及其生物合成分析

Discovery and biosynthetic analysis of skyllamycins from Streptomyces pratensis SCSIO LCY05 derived from Ascidian

目的挖掘海鞘来源放线菌Streptomyces pratensis SCSIO LCY05生产含肉桂酰独特结构单元的skyllamycins类环肽的潜能,并深入分析skyllamycins生物合成基因簇的新特征。方法利用Illumina Hiseq和Pacbio SMRT测序平台对S.pratensis SCSIO LCY05基因组DNA进行全基因组测序,通过生物信息学手段对菌株基因组的生物合成基因簇进行预测和基因功能注释,采用OSMAC(one strain many compounds)策略对S.pratensis SCSIO LCY05菌株进行发酵优化,结合萃取法、色谱学和波谱学等方法对该菌株的次级代谢产物进行分离和结构鉴定,并对得到的化合物的生物合成途径进行推导及对新颖的合成特征进行挖掘。结果全基因组测序结果表明,S.pratensis SCSIO LCY05菌株的基因组全长为8.42 Mbp,共含有35个生物合成基因簇,该基因组上的基因簇20与skyllamycins生物合成基因簇的相似度为95%。在OSMAC策略的优化基础上,发现S.pratensis SCSIO LCY05菌株在SCAS培养基中出现了2个具有典型吸收特征的峰,经鉴定为skyllamycin A和skyllamycin B环肽化合物,并推导了其生物合成途径。进一步的聚类分析发现skyllamycins装配线上的C_(11)结构域可能是具有缩合和异构化双重功能的结构域,负责skyllamycins中第11个氨基酸的组装和异构化。结论本研究不仅发现海鞘来源放线菌S.pratensis SCSIO LCY05经OSMAC策略的优化后可产生skyllamycins类环肽化合物,并揭示了skyllamycins生物合成过程中的新特征,同时也为skyllamycins类化合物生物合成机制的深入阐释及其进一步的开发利用提供了新的菌株资源。

Objective To investigate the potential of cinnamoyl structural unit containing skyllamycins production in Streptomyces pratensis SCSIO LCY05 derived from Ascidian and analyze the new characteristics of skyllamycins biosynthetic gene cluster(skl BGC).Methods The whole genome of S.pratensis SCSIO LCY05 was sequenced using the Illumina Hiseq and PacBio SMRT platform,and the biosynthetic gene clusters of the secondary meatbolites were predicted and the functions of ORFs in the biosynthetic gene cluster were annotated by online bioinformatic softwares.The one strain many compounds(OSMAC)strategy was used to conduct the fermentation optimization of S.pratensis SCSIO LCY05.The secondary metabolites of the strain were isolated and structurally characterized by extraction,chromatography and spectroscopic data analyses,etc.The biosynthetic pathway and novel biosynthetic characteristics of the obtained compounds were analyzed.Results The whole genome sequencing results showed that the genome size of S.pratensis SCSIO LCY05 was 8.42 Mbp,containing a total of 35 biosynthetic gene clusters.The 20 th gene cluster on the genome was 95%similar to the reported skyllamycins biosynthetic gene cluster and predicted to charge for the biosynthesis of cinnamyol unit containing natural products in S.pratensis SCSIO LCY05.Based on the OSMAC strategy,two peaks with typical absorption characteristics were found in SCAS medium of S.pratensis SCSIO LCY05,which were identified as skyllamycin A and skyllamycin B.The biosynthetic pathway of skyllamycin A and B was analyzed in detail.Further sequence alignment indicated that the C_(11) domain on skyllamycin assembly line was a special domain with dual functions that responsible for the condensation and epimerization of the 11 th amino acid in skyllamycin.Concluison In this study,we found that the cyclic peptides skyllamycins could be produced in S.pratensis SCSIO LCY05 by OSMAC strategy,and discovered new characteristics in its biosynthetic assembly line.Notably,this study also provided a new strain resource for the indepth elucidation of the biosynthetic mechanism of skyllamycins and subsequent development and utilization.