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<sup>*</sup>

doi:10.13523/j.cb.2104040

中国生物工程杂志

2021, Vol. 41

Issue (9): 37-47 DOI: 10.13523/j.cb.2104040

研究报告

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^*

陈亚超¹,李楠楠¹,刘子迪¹,胡冰^1,^**(

),李春²

1 北京理工大学化学与化工学院化学工程系生物化工研究所北京 100081
2 清华大学工业生物催化教育部重点实验室北京 100084

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

CHEN Ya-chao¹,LI Nan-nan¹,LIU Zi-di¹,HU Bing^1,^**(

),LI Chun²

1 Institute of Biochemical Engineering, Department of Chemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, China
2 Key Lab for Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China

全文: PDF(2015 KB) HTML

摘要：

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。

关键词： 甘草内生菌; 宏基因组测序; 微生物多样性; 甘草酸; 功能基因

Abstract:

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.

Key words: Endophytes of licorice Metagenomic sequencing Microbial diversity Glycyrrhizin Functional gene

收稿日期: 2021-04-23 出版日期: 2021-09-30

ZTFLH:

Q812

基金资助: * 国家自然科学基金(21736002);国家重点研发计划(2018YFA0901800);国家重点研发计划(2020YFA0908300)

通讯作者: 胡冰 E-mail: binghu319@bit.edu.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	陈亚超
	李楠楠
	刘子迪
	胡冰
	李春

引用本文:

陈亚超,李楠楠,刘子迪,胡冰,李春. 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^*[J]. 中国生物工程杂志, 2021, 41(9): 37-47.

CHEN Ya-chao,LI Nan-nan,LIU Zi-di,HU Bing,LI Chun. Metagenomic Mining of Functional Genes Related to Glycyrrhizin Synthesis from Endophytes of Licorice. China Biotechnology, 2021, 41(9): 37-47.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2104040 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I9/37

图1 甘草酸的生物合成途径

表1 宏基因组数据总览

图2 甘草内生菌在门(a)、属(b)和种(c)水平上的群落丰度百分比

表2 COG功能注释分析能量、氨基酸、细胞修复、离子转运相关基因的绝对丰度

图3 KEGG功能注释分析

表3 碳水化合物活性酶注释与基因丰度总览

图4 可能参与到甘草三萜合成的P450s在总P450s中的占比

图5 甘草内生菌UGTs与其他UGTs的系统发育分析

[1]	Nomura Y, Seki H, Suzuki T, et al. Functional specialization of UDP -glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin. The Plant Journal, 2019, 99(6):1127-1143. doi: 10.1111/tpj.14409 pmid: 31095780
[2]	Zhao Y J, Lv B, Feng X D, et al. Perspective on biotransformation and de novo biosynthesis of licorice constituents. Journal of Agricultural and Food Chemistry, 2017, 65(51):11147-11156. doi: 10.1021/acs.jafc.7b04470
[3]	Zhang L, Ren S C, Liu X F, et al. Mining of UDP-glucosyltrfansferases in licorice for controllable glycosylation of pentacyclic triterpenoids. Biotechnology and Bioengineering, 2020, 117(12):3651-3663. doi: 10.1002/bit.v117.12
[4]	Matsui S, Matsumoto H, Sonoda Y, et al. Glycyrrhizin and related compounds down-regulate production of inflammatory chemokines IL-8 and eotaxin 1 in a human lung fibroblast cell line. International Immunopharmacology, 2004, 4(13):1633-1644. doi: 10.1016/j.intimp.2004.07.023
[5]	Huang R Y, Chu Y L, Jiang Z B, et al. Glycyrrhizin suppresses lung adenocarcinoma cell growth through inhibition of thromboxane synthase. Cellular Physiology and Biochemistry, 2014, 33(2):375-388. doi: 10.1159/000356677
[6]	Cinatl J, Morgenstern B, Bauer G, et al. Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. The Lancet, 2003, 361(9374):2045-2046. doi: 10.1016/S0140-6736(03)13615-X
[7]	Murck H. Symptomatic protective action of glycyrrhizin (licorice) in COVID-19 infection? Frontiers in Immunology, 2020, 11:1239. DOI: 10.3389/fimmu.2020.01239. doi: 10.3389/fimmu.2020.01239
[8]	Bailly C, Vergoten G. Glycyrrhizin: an alternative drug for the treatment of COVID-19 infection and the associated respiratory syndrome? Pharmacology & Therapeutics, 2020, 214:107618.
[9]	Sun W T, Qin L, Xue H J, et al. Novel trends for producing plant triterpenoids in yeast. Critical Reviews in Biotechnology, 2019, 39(5):618-632. doi: 10.1080/07388551.2019.1608503
[10]	Wang W, He P, Zhao D D, et al. Construction of Escherichia coli cell factories for crocin biosynthesis. Microbial Cell Factories, 2019, 18(1):1-11. doi: 10.1186/s12934-018-1049-x
[11]	Gupta S, Chaturvedi P, Kulkarni M G, et al. A critical review on exploiting the pharmaceutical potential of plant endophytic fungi. Biotechnology Advances, 2020, 39:107462. doi: 10.1016/j.biotechadv.2019.107462
[12]	Chen Y C, Hu B, Xing J M, et al. Endophytes: the novel sources for plant terpenoid biosynthesis. Applied Microbiology and Biotechnology, 2021, 105(11):4501-4513. doi: 10.1007/s00253-021-11350-7
[13]	陈静, 许贞, 张雪, 等. 不同产地甘草内生真菌多样性及分离条件研究. 药学学报, 2019, 54(2):373-379.
	Chen J, Xu Z, Zhang X, et al. Diversity and isolation parameters of endophytes from Glycyrrhiza uralensis of different habitats. Acta Pharmaceutica Sinica, 2019, 54(2):373-379.
[14]	Datta S, Rajnish K N, Samuel M S, et al. Metagenomic applications in microbial diversity, bioremediation, pollution monitoring, enzyme and drug discovery. A review. Environmental Chemistry Letters, 2020, 18(4):1229-1241. doi: 10.1007/s10311-020-01010-z
[15]	Caparco A A, Pelletier E, Petit J L, et al. Metagenomic mining for amine dehydrogenase discovery. Advanced Synthesis & Catalysis, 2020, 362(12):2427-2436.
[16]	Carrión V J, Perez-Jaramillo J, Cordovez V, et al. Pathogen-induced activation of disease-suppressive functions in the endophytic root microbiome. Science, 2019, 366(6465):606-612. doi: 10.1126/science.aaw9285 pmid: 31672892
[17]	Marshall J R, Yao P Y, Montgomery S L, et al. Screening and characterization of a diverse panel of metagenomic imine reductases for biocatalytic reductive amination. Nature Chemistry, 2021, 13(2):140-148. doi: 10.1038/s41557-020-00606-w pmid: 33380742
[18]	Mochida K, Sakurai T, Seki H, et al. Draft genome assembly and annotation of Glycyrrhiza uralensis, a medicinal legume. The Plant Journal, 2017, 89(2):181-194. doi: 10.1111/tpj.13385
[19]	Kergourlay G, Taminiau B, Daube G, et al. Metagenomic insights into the dynamics of microbial communities in food. International Journal of Food Microbiology, 2015, 213:31-39. doi: 10.1016/j.ijfoodmicro.2015.09.010 pmid: 26414193
[20]	宋素琴, 欧提库尔·玛合木提, 张志东, 等. 新疆胀果甘草内生菌的分离和鉴定. 微生物学通报, 2007, 34(5):867-870.
	Song S Q, Otkur M, Zhang Z D, et al. Isolation and characterization of endophytic microorganisms in glaycyrrhiza inflat bat. from Xinjiang. Microbiology, 2007, 34(5):867-870.
[21]	李文军, 郑素慧, 毛培宏, 等. 新疆胀果甘草内生菌的分离及产甘草酸菌株的筛选. 生物技术, 2008, 18(5):28-31.
	Li W J, Zheng S H, Mao P H, et al. Isolation of endophyte from Glycyrrhiza inflata batalin of Xinjiang uygur autonomous region and screening of glycyrrhizic acid-producing strain. Biotechnology, 2008, 18(5):28-31.
[22]	Tiwari P, Sangwan R S, Sangwan N S. Plant secondary metabolism linked glycosyltransferases: an update on expanding knowledge and Scopes. Biotechnology Advances, 2016, 34(5):714-739. doi: 10.1016/j.biotechadv.2016.03.006
[23]	Girvan H M, Munro A W. Applications of microbial cytochrome P450 enzymes in biotechnology and synthetic biology. Current Opinion in Chemical Biology, 2016, 31:136-145. doi: 10.1016/j.cbpa.2016.02.018 pmid: 27015292
[24]	Fiallos-Jurado J, Pollier J, Moses T, et al. Saponin determination, expression analysis and functional characterization of saponin biosynthetic genes in Chenopodium quinoa leaves. Plant Science, 2016, 250:188-197. doi: S0168-9452(16)30094-2 pmid: 27457995
[25]	Morita M, Shibuya M, Kushiro T, et al. Molecular cloning and functional expression of triterpene synthases from pea (Pisum sativum). European Journal of Biochemistry, 2000, 267(12):3453-3460. pmid: 10848960
[26]	Hayashi H, Huang P Y, Kirakosyan A, et al. Cloning and characterization of a cDNA encoding β-amyrin synthase involved in glycyrrhizin and soyasaponin biosyntheses in licorice. Biological and Pharmaceutical Bulletin, 2001, 24(8):912-916. pmid: 11510484
[27]	Iturbe-Ormaetxe I, Haralampidis K, Papadopoulou K, et al. Molecular cloning and characterization of triterpene synthases from Medicago truncatula and Lotus japonicus. Plant Molecular Biology. 2003, 51(5):731-743. pmid: 12683345
[28]	Zhu M, Wang C X, Sun W T, et al. Boosting 11-oxo-β-amyrin and glycyrrhetinic acid synthesis in Saccharomyces cerevisiae via pairing novel oxidation and reduction system from legume plants. Metabolic Engineering, 2018, 45:43-50. doi: 10.1016/j.ymben.2017.11.009
[29]	Wilson A E, Tian L. Phylogenomic analysis of UDP-dependent glycosyltransferases provides insights into the evolutionary landscape of glycosylation in plant metabolism. The Plant Journal, 2019, 100(6):1273-1288. doi: 10.1111/tpj.v100.6
[30]	Brazier-Hicks M, Gershater M, Dixon D, et al. Substrate specificity and safener inducibility of the plant UDP-glucose-dependent family 1 glycosyltransferase super-family. Plant Biotechnology Journal, 2018, 16(1):337-348. doi: 10.1111/pbi.12775 pmid: 28640934
[31]	Chen K, Hu Z M, Song W, et al. Diversity of O-glycosyltransferases contributes to the biosynthesis of flavonoid and triterpenoid glycosides in Glycyrrhiza uralensis. ACS Synthetic Biology, 2019, 8(8):1858-1866. doi: 10.1021/acssynbio.9b00171
[32]	Tiwari P, Bae H H. Horizontal gene transfer and endophytes: an implication for the acquisition of novel traits. Plants, 2020, 9(3):305. doi: 10.3390/plants9030305
[33]	Tiwari R, Nain L, Labrou N E, et al. Bioprospecting of functional cellulases from metagenome for second generation biofuel production: a review. Critical Reviews in Microbiology, 2018, 44(2):244-257. doi: 10.1080/1040841X.2017.1337713
[34]	Xin Y P, Guo T T, Zhang Y, et al. A new β-galactosidase extracted from the infant feces with high hydrolytic and transgalactosylation activity. Applied Microbiology and Biotechnology, 2019, 103(20):8439-8448. doi: 10.1007/s00253-019-10092-x

[1]	安云鹤, 程小艳, 田彦捷, 马凯, 高丽娟, 武会娟. DNA提取对微生物多样性测序分析的影响[J]. 中国生物工程杂志, 2017, 37(11): 12-18.
[2]	吴梦, 刘作华, 林保忠, 兰国成, 邹贤刚, 葛良鹏. 转基因猪研究进展[J]. 中国生物工程杂志, 2015, 35(3): 92-98.
[3]	王世伟, 王卿惠, 翟丽萍, 刘军, 郑苗苗, 王芳, 于志丹. 原核微生物腈转化酶研究进展[J]. 中国生物工程杂志, 2015, 35(10): 100-107.
[4]	杨毅, 李治, 高玲霞, 孙燕. 荧光假单胞菌抗生性代谢产物合成相关基因的研究现状[J]. 中国生物工程杂志, 2012, 32(08): 100-106.
[5]	刘菊华, 徐碧玉, 张建平, 贾彩红, 王甲水, 张建斌, 金志强. 香蕉基因组测序及胁迫相关功能基因研究进展[J]. 中国生物工程杂志, 2012, 32(03): 110-114.
[6]	王世伟, 王敏. 腈类物降解菌多样性和产腈水合酶研究进展[J]. 中国生物工程杂志, 2011, 31(9): 117-123.
[7]	刘红涛,冯书营,陈涛,薛乐勋. 杜氏盐藻分子生物学最新进展及展望[J]. 中国生物工程杂志, 2007, 27(10): 113-118.
[8]	张莹,杨耀武,王健伟,,屈建国,洪涛. RNA干扰文库在功能基因组学研究中的发展及应用[J]. 中国生物工程杂志, 2006, 26(07): 84-89.
[9]	信吉阁,曾养志,韩佃刚,王晓洪. 功能基因组学及其研究进展[J]. 中国生物工程杂志, 2006, 26(0): 162-165.
[10]	庄金秋, 杨丽梅, 贾杏林. 功能基因组学研究概述[J]. 中国生物工程杂志, 2005, 25(S1): 204-209.
[11]	郑卉, 李良智, 葛志强, 元英进. 代谢物组学及其在微生物研究中的应用[J]. 中国生物工程杂志, 2005, 25(5): 6-9.
[12]	谭彩霞, 张亚芳, 陈宗祥, 殷跃军, 纪雪梅, 杨勇, 潘学彪. 两个抗水稻纹枯病主效数量基因的鉴定[J]. 中国生物工程杂志, 2004, 24(4): 79-80.
[13]	朱平, 王伟, 程克棣. 药用植物功能基因[J]. 中国生物工程杂志, 2004, 24(2): 3-8.
[14]	周晓馥, 肖乃仲, 白云峰, 王兴智. 植物功能基因组学的研究策略[J]. 中国生物工程杂志, 2002, 22(6): 13-17.
[15]	贺超英, 陈受宜. 基因组学方法在植物抗逆性研究中的应用[J]. 中国生物工程杂志, 2001, 21(1): 29-32.

Viewed

Full text

Abstract

Cited

Shared

Discussed