一个紫芝多产物倍半萜合酶的功能鉴定

褚丽华; 王丽芝; 陈士林; 曾欣宜; 徐江; 李滢; 孙超

doi:10.13422/j.cnki.syfjx.20191216

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一个紫芝多产物倍半萜合酶的功能鉴定

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- 一个紫芝多产物倍半萜合酶的功能鉴定
- Functional Identification of a Multi-product Sesquiterpene Synthase from Ganoderma sinense
- 中国实验方剂学杂志 2019年25卷第12期页码：151-157
- 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：
  
  褚丽华，在读硕士，从事药用植物功能基因组学研究，Tel：010-57833197，E-mail：chulihua0427@163.com
  孙超，研究员，从事药用植物功能基因组学研究，Tel：010-57833197，E-mail：csun@implad.ac.cn
- 基金信息：
  
  国家自然科学基金项目(81573704)
- DOI：10.13422/j.cnki.syfjx.20191216
  中图分类号： R284.1; R2-031; R22; R289
- 收稿日期：2019-03-25，
  
  网络出版日期：2019-04-19，
  
  纸质出版日期：2019-06-20
- 稿件说明：
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褚丽华, 王丽芝, 陈士林, 等. 一个紫芝多产物倍半萜合酶的功能鉴定[J]. 中国实验方剂学杂志, 2019,25(12):151-157.

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.
褚丽华, 王丽芝, 陈士林, 等. 一个紫芝多产物倍半萜合酶的功能鉴定[J]. 中国实验方剂学杂志, 2019,25(12):151-157. DOI： 10.13422/j.cnki.syfjx.20191216.

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

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