Functional Identification of a Multi-product Sesquiterpene Synthase from <italic style="font-style: italic">Ganoderma sinense</italic>

Li-hua CHU; Li-zhi WANG; Shi-lin CHEN; Xin-yi ZENG; Jiang XU; Ying LI; Chao SUN

doi:10.13422/j.cnki.syfjx.20191216

您当前的位置：

首页 >

文章列表页 >

Functional Identification of a Multi-product Sesquiterpene Synthase from Ganoderma sinense

Resource and Quality Evaluation | 更新时间：2021-02-09

- Functional Identification of a Multi-product Sesquiterpene Synthase from Ganoderma sinense
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 12, Pages: 151-157(2019)
- 作者机构：
  
  1.中国医学科学院&北京协和医学院　药用植物研究所，北京　 100193；
  2.天津中医药大学　中药学院，天津　 300193；
  3.中国中医科学院　中药研究所，北京　 100700
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20191216
  CLC： R284.1; R2-031; R22; R289
- Received：25 March 2019，
  
  Published Online：19 April 2019，
  
  Published：20 June 2019
- 稿件说明：
移动端阅览
Li-hua CHU, Li-zhi WANG, Shi-lin CHEN, et al. Functional Identification of a Multi-product Sesquiterpene Synthase from Ganoderma sinense[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(12): 151-157.
DOI：

Li-hua CHU, Li-zhi WANG, Shi-lin CHEN, et al. Functional Identification of a Multi-product Sesquiterpene Synthase from Ganoderma sinense[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(12): 151-157. DOI： 10.13422/j.cnki.syfjx.20191216.

摘要

目的：

紫芝是我国最著名的药用真菌之一，作为担子菌亚门的模式药用真菌，其倍半萜合酶的功能鉴定对真菌倍半萜的生物合成与调控研究具有重要意义。

方法：

通过挖掘紫芝基因组数据发现一个灵芝倍半萜合酶基因，将其与已经鉴定的两个紫芝倍半萜合酶和非冗余蛋白数据库(Nr数据库)中与其同源性最高的3个萜类合酶进行多重序列比对分析其保守结构域。然后，在大肠埃希菌中进行异源表达，通过SDS-PAGE对其蛋白表达情况进行检测。利用固相微萃取收集挥发性物质，并通过GC-MS对其产物进行检测。

结果：

该酶含有倍半萜保守基序DDXXDE和NSE/DTE，可在大肠埃希菌中可溶性表达，GC-MS分析发现，该酶可以内源性法尼基焦磷酸(FPP)为底物合成11种倍半萜类化合物，出峰时间为18.6 min的产物

-cadinol是主要产物，已有研究表明

-cadinol具有明显的抗癌活性。通过与对照品比对进一步确定其中3个产物分别是

-muurlene，

-muurolene和

-cadinene。

结论：

紫芝多产物倍半萜合酶的功能鉴定可推动其产物多样性的形成机制研究，为通过酶工程技术改良酶的催化活性生产稀有或新型倍半萜类化合物奠定基础。

Abstract

Objective:

Ganoderma sinense

is one of the most famous medicinal fungi in China. Because it is a model medicinal fungus of basidiomycete

the functional identification of its sesquiterpene synthase is of great significance for the biosynthesis and regulation studies of fungal sesquiterpene.

Method:

A sesquiterpene synthase gene was discovered by mining the genome data of

sinense

. The sesquiterpene's conservative motifs was analyzed through multiple sequence alignment with two identified sesquiterpene synthases of

sinense

and three terpenoid synthases in the Nr database

which have the highest similarity to it. Subsequently

heterologous expression was observed in

Escherichia coli

and protein expression was detected by SDS-PAGE. Volatile compounds were collected by solid phage microextraction (SPME) and detected by GC-MS.

Result:

The enzyme containing sesquiterpene conserved motifs DDXXDE and NSE/DTE were efficiently expressed in

coli

and 11 sesquiterpenes were synthesized with endogenous FPP as the substrate. The product

-cadinol at 18.6 min was considered to be the main product

and previous studies showed a significant anticancer activity. According to the comparison with chemical standards

three products were identified as

-muurlene

-muurolene and

-cadinene

respectively.

Conclusion:

The functional identification of multi-product sesquiterpene synthase from

sinense

can promote the study on the mechanism underlying its product diversity

and lay a foundation for the production of rare or novel sesquiterpenes by improving the catalytic activity of enzymes with enzyme engineering technology.

关键词

Keywords

references

ZHU Y J , XU J , SUN C , et al . Chromosome-level genome map provides insights into diverse defense mechanisms in the medicinal fungus Ganoderma sinense [J]. Sci Rep , 2015 , 5 : 11087 .

国家药典委员会．中华人民共和国药典．一部 [M]．北京：中国医药科技出版社， 2015 ： 188 ．

杜琳，刘玉容，叶峻，等．紫芝中三萜类化学成分研究 [J]．天然产物研究与开发， 2018 ， 30 ( 10 )： 1669 - 1673 ．

陈若芸，于德泉．灵芝三萜化学成分研究进展 [J]．药学学报， 1990 ， 25 ( 12 )： 940 - 953 ．

LIU J Q , WANG C F , LI Y , et al . Isolation and bioactivity evaluation of terpenoids from the medicinal fungus Ganoderma sinense [J]. Planta Med , 2012 , 78 ( 4 ): 368 - 376 .

Wawrzyn G T , Quin M B , Choudhary S , et al . Draft genome of Omphalotus olearius provides a predictive framework for sesquiterpenoid natural product biosynthesis in basidiomycota [J]. Chem Biol , 2012 , 19 ( 6 ): 772 - 783 .

Agger S , Lopez-Gallego F , Schmidt-Dannert C . Diversity of sesquiterpene synthases in the basidiomycete Coprinus cinereus [J]. Mol Microbiol , 2009 , 72 ( 5 ): 1181 - 1195 .

Ichinose H , Kitaoka T . Insight into metabolic diversity of the brown-rot basidiomycete Postia placenta responsible for sesquiterpene biosynthesis: semi-comprehensive screening of cytochrome P450 monooxygenase involved in protoilludene metabolism [J]. Microb Biotechnol , 2018 , 11 ( 5 ): 952 - 965 .

Engels B , Heinig U , Grothe T , et al . Cloning and characterization of an Armillaria gallica cDNA encoding protoilludene synthase, which catalyzes the fifirst committed step in the synthesis of antimicrobial melleolides [J]. J Biol Chem , 2011 , 286 ( 9 ): 6871 - 6878 .

王丽芝，王海英，浦香东，等．基于紫芝基因组的倍半萜合酶基因的发掘与功能鉴定 [J]．中国科学：生命科学， 2018 ， 48 ( 4 )： 447 - 454 ．

Quin M B , Flynn C M , Schmidt-Dannert C . Traversing the fungal terpenome [J]. Nat Prod Rep , 2014 , 31 ( 10 ): 1449 - 1473 .

LU J J , DANG Y Y , HUANG M , et al . Anti-cancer properties of terpenoids isolated from Rhizoma Curumae-a review [J]. J Ethnopharmacol , 2012 , 143 ( 2 ): 406 - 411 .

Yap H Y , MuriaGonzalez M J , Kong B H , et al . Heterologous expression of cytotoxic sesquiterpenoids from the medicinal mushroom Lignosus rhinocerotis in yeast [J]. Microb Cell Fact , 2017 , 16 ( 1 ): 103 .

Dibwe D F , SUN S J , Ueda Junya , et al . Discovery of potential antiausterity agents from the Japanese cypress Chamaecyparis obtusa [J]. Bioorg Med Chem Lett , 2017 , 27 ( 21 ): 4898 - 4903 .

Masao T , Akemi U , Shigenobu A . T-cadinol and calamenene induce dendritic cells from human monocytes and drive Th1 polarization [J]. Eur J Pharmacol , 2006 , 537 ( 1/3 ): 190 - 199 .

Miller D J , Allemann R K . Sesuiterpene synthases: passive catalysts or active players [J]. Nat Pro Rep , 2012 , 29 ( 1 ): 60 - 71 .

Degenhardt J , Köllner T G , Gershenzon J . Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants [J]. Phytochem , 2009 , 70 ( 15/16 ): 1621 - 1637 .

Gambliel H , Croteau R . Pinene cyclases Ⅰ and Ⅱ. Two enzymes from sage (Salvia officinalis) which catalyze stereospecific cyclizations of geranyl pyrophosphate to monoterpene olefins of opposite configuration [J]. J Biol Chem , 1984 , 259 : 740 - 748 .

Steele C L , Crock J , Bohlmann J , et al . Sesquiterpene synthases from grand fir (Abies grandis). Comparison of constitutive and wound-induced activities, and cDNA isolation, characterization, and bacterial expression of delta-selinene synthase and gamma-humulene synthase [J]. J Biol Chem , 1998 , 273 ( 4 ): 2078 - 2089 .

Vattekkatte A , Garms S , Brandt W , et al . Enhanced structural diversity in terpenoid biosynthesis: enzymes, substrates and cofactors [J]. Org Biomol Chem , 2018 , 16 ( 3 ): 348 - 362 .

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Effect of Naozhenning Granules on Energy Metabolism of Cortical Mitochondria in Rat Model of Post-concussion Syndrome

Biosynthesis of Steroidal Saponins in Herbs

Effect and Mechanism of Arbuscular Mycorrhizal Fungi in Herbs

Advances in Biosynthesis and Regulation Mechanism of Notoginseng Saponins

Related Author

ZHAO Le

GAO Li

WANG Yonghui

ZHANG Weiyi

LIU Qizhao

WANG Tiantian

WU Liya

WEI Nannan

Related Institution

Shanxi University of Chinese Medicine

School of Traditional Chinese Medicine， Yunnan University of Chinese Medicine

AI问答

Postal code：100700
Tel：010-84076882 Email：syfjx_2010@188.com
Technical support is provided by Beijing Founder electronics co., LTD 京ICP备11006657号-10 京公网安备11010802024621
It is recommended to read the content of this site in Chrome&IE9+. Please switch to extreme mode in browser 360.
Cookies We use cookies to help provide and enhance our service and tailor content. By continuing, you agree to the use of cookies.

⁰

Functional Identification of a Multi-product Sesquiterpene Synthase from Ganoderma sinense

DOI：10.13422/j.cnki.syfjx.20191216

摘要

Abstract

关键词

Keywords

references