源于甘草内生菌的甘草酸合成相关功能基因的宏基因组挖掘

Metagenomic Mining of Functional Genes Related to Glycyrrhizin Synthesis from Endophytes of Licorice

目的:甘草作为一种传统中药被广泛使用。甘草酸是甘草中的主要活性成分,为五环三萜类化合物,具有抗炎、抗病毒、肝保护等多种药理作用,而且近两年已被用于新型冠状病毒肺炎(COVID-19)的临床治疗。随着代谢工程与合成生物学技术的发展,人们逐渐实现了甘草酸及其前体物的微生物合成。但由于植物基因与微生物底盘不适配等原因,已报道的生物合成法产量较低。近些年,研究者发现植物内生菌拥有丰富的功能特性,可作为植物活性产物合成的潜在资源,在代谢工程领域,具有重要的研究价值及巨大的市场应用前景。因此,拟对甘草内生菌群落进行深入研究,挖掘可用于甘草酸合成的微生物源功能基因。方法:从新疆塔城市额敏县采集了三年生乌拉尔甘草的主根样品,进行了内生菌群落的宏基因组测序,对数据进行了内生菌的群落结构及功能基因多样性分析,并通过功能基因注释与系统发育树分析,挖掘了可能参与甘草酸合成代谢的功能基因。结果:通过对群落结构进行丰度分析,发现甘草根部样品中的优势内生菌菌种为反硝化类固醇杆菌(Steroidobacter denitrificans)、苯丙酸杆菌(Phenylobacterium zucineum)、未分类苯基杆菌(unclassified Phenylobacterium)、苯基杆菌(Phenylobacterium sp.)等;通过COG数据库、KEGG数据库和CAZy数据库对其功能基因进行注释,发现在甘草内生菌中含有123个细胞色素P450(cytochrome P450,CYP450)及520个UDP-糖基转移酶(UGT)编码基因。结论:首次利用宏基因组测序与分析方法来了解乌拉尔甘草内生菌群落结构与功能基因组成,并证明了甘草内生群落中含有丰富的细胞色素P450及UGT编码基因,为后续全面、深入研究甘草内生菌的生物学功能,以及它们如何转变为甘草酸生物合成资源,奠定了理论基础。

Objective:Licorice is widely used as a traditional Chinese medicine.Glycyrrhizin is the main active ingredient in licorice,a pentacyclic trterpenoid compound,has many pharmaceutical functions,such as anti-inflammatory,antiviral,and liver protection,and has been used in the clinical treatment of COVID-19.With the development of metabolic engineering and synthetic biology,the microbial synthesis of glycyrrhizin and its precursors has been gradually realized.However,the yield is low due to the incompatibility between plant genes and microbial chassis.In last decades,endophytes have been reported to harbor a wealth of functional traits,and could be directly or indirectly used for the production of plant-derived active compounds,indicating that investigation on endophytes has great academic potentials and application values in the field of metabolic engineering.Therefore,this study intends to conduct an in-depth study on the endophyte community of licorice and dig out the microbial-source functional genes that can be used for glycyrrhizin synthesis.Methods:In this study,three year old main root samples of Glycyrrhiza uralensis were collected from Emin County,Tacheng City,Xinjiang Uygur Autonomous Reyion.Metagenomic sequencing of endophytic communities was carried out,and the community structure and functional gene diversity of endophytic communities were analyzed.Through functional gene annotation and phylogenetic analysis,functional genes that may be involved in glycyrrhizin synthesis were excavated.Results:By analyzing the abundance of the community structure,it was found that the dominant endophytes in the licorice root were Steroidobacter denitrificans,Phenylobacterium zucineum,unclassified Phenylobacterium,and Phenylobacterium sp..The metagenomic data were functionally annotated by COG database,KEGG database,and CAZy database,and the endophytic genes encoding enzymes involved in glycyrrhizin synthetic pathway,such as cytochrome P450 and UDP-glycosyltransferase,were found to be abundant in the endophytes of licorice.Conclusion:This study was the first to use metagenomic sequencing and analysis methods to understand the community structure and functional gene composition of endophytes from G.uralensis.It was proved that the endophytic community contained abundant cytochrome P450 and UGT coding genes,which laid a theoretical foundation for further comprehensive and in-depth study of the biological functions of endophytes from G.uralensis and how they transform into glycyrrhizin biosynthesis resources.