高效合成麦角硫因的蛹虫草工程菌株构建

doi:10.13560/j.cnki.biotech.bull.1985.2026-0314

摘要/Abstract

摘要：

目的 Egt1和Egt2是麦角硫因（ergothioneine）生物合成途径中的关键酶。通过在蛹虫草（Cordyceps militaris）中过表达麦角硫因生物合成基因Egt1和Egt2，构建高效合成麦角硫因的工程菌株。方法以蛹虫草尿苷/尿嘧啶和组氨酸双营养缺陷型菌株CmΔpyrGΔhisB为背景菌株，通过农杆菌转化法（Agrobacterium tumefaciens-mediated transformation, ATMT）在蛹虫草中过表达内源和异源麦角硫因合成途径的关键基因，并测定转基因蛹虫草中麦角硫因和虫草素含量。结果蛹虫草内源基因过表达菌株的麦角硫因含量提高至7.62 mg/g，是野生型菌株的14倍；同时虫草素的含量提高至6.81 mg/g，约为野生型菌株的5倍。异源过表达菌株中麦角硫因的产量进一步提高到8.45 mg/g，是野生型菌株的15倍。此外，添加前体甲硫氨酸不能有效提高异源过表达菌株中麦角硫因的含量。结论利用农杆菌转化法构建蛹虫草工程菌株可实现麦角硫因和虫草素高效协同生产。

关键词: 蛹虫草, 农杆菌转化, 丝状真菌, 麦角硫因, 虫草素

Abstract:

Objective Egt1 and Egt2 are key enzymes in the ergothioneine biosynthesis pathway. This study aims to construct high-yielding Cordyceps militaris strains by overexpressing key genes (Egt1 and Egt2) involved in the ergothioneine biosynthetic pathway. Method The dual uridine/uracil and histidine auxotrophic strain CmΔpyrGΔhisB was used as the parental strain. Agrobacterium tumefaciens-mediated transformation (ATMT) method was employed to overexpress both endogenous and heterologous ergothioneine biosynthetic genes in C. militaris. The contents of ergothioneine and cordycepin in the transgenic strains were subsequently determined. Result Overexpression of endogenous genes increased ergothioneine content to 7.62 mg/g, representing a 14‑fold increase over the wild-type strain; meanwhile, the cordycepin content reached 6.81 mg/g, approximately 5‑fold that of the wild‑type strain. Co-expression of heterologous genes NcEgt1 and NcEgt2 further enhanced the ergothioneine yield to 8.45 mg/g, representing a 15‑fold increase over the wild‑type strain. Moreover, precursor methionine supplementation did not significantly enhance the ergothioneine content in heterologous overexpressed strains. Conclusion The engineered C. militaris strains constructed via ATMT method enable the highly efficient and synergistic co‑production of both ergothioneine and cordycepin.

Key words: Cordyceps militaris, Agrobacterium tumefaciens-mediated transformation, filamentous fungus, ergothioneine, cordycepin

王丽红, 崔璐瑶, 薛萍红, 邓运鸿, 高睿, 胡志宏. 高效合成麦角硫因的蛹虫草工程菌株构建[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0314.

WANG Li-hong, CUI Lu-yao, XUE Ping-hong, DENG Yun-hong, GAO Rui, HU Zhi-hong. Construction of an Engineered Cordyceps militaris Strain for Efficient Ergothioneine Synthesis[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0314.

图/表 11

图1 麦角硫因真菌生物合成途径

Fig. 1 Fungal biosynthetic pathway of ergothioneine

表1 本研究所用生物信息学软件

Table 1 Bioinformatics software used in this study

分析软件 Analysis software	用途 Application	网址 Web site
NCBI-Blast	基因同源性分析Gene homology analysis	https：//blast.ncbi.nlm.nih.gov/Blast.cgi
SignalP 4.1	信号肽预测Prediction of signal peptide	https：//services.healthtech.dtu.dk/service.php SignalP-4.1
PSORT Ⅱ	亚细胞定位Subcellular localization	https：//www.genscript.com/psort.html
MEGA 11.0	系统进化树分析Phylogenetic analysis	https：//www.megasoftware.net/
TBtools-Ⅱ	绘制基因结构Gene structure drawing	https：//github.com/CJ-Chen/TBtools-Ⅱ/releases

表2 构建载体所用引物

Table 2 Primers used for vector construction

引物名称 Primer name	引物序列 Primer sequence （5΄-3΄）	大小 Size （bp）
IF-pEX2D-CmEgt1-DsRed-F	CAAACACCTTCAAACACGTGATGCCTGCCGTCAAGGAGGC	3 241
IF-pEX2D-CmEgt1-DsRed-R	ATGATATCCTTAAGCACGTGAGCGACATCGCGCACAAGGC	3 241
IF-pEX1-CmEgt2-GFP-F	CTCGAGTACGTAGGTACCATGACCGTTACCAATCAGAC	1 371
IF-pEX1-CmEgt2-GFP-R	CCCTTGCTCACCATGGTACCAATCTTATACTCCCCATTGG	1 371
IF-pEX2D-NcEgt1-DsRed-F	CAAACACCTTCAAACACGTGATGCCGAGTGCCGAATCCAT	2 631
IF-pEX2D-NcEgt1-DsRed-R	ATGATATCCTTAAGCACGTGCAAATCCCTAACAACTCTCG	2 631
IF-pEX1-NcEgt2-GFP-F	CTCGAGTACGTAGGTACCATGGTCGCCACCACCGTC	1 422
IF-pEX1-NcEgt2-GFP-R	CTTGCTCACCATGGTACCGGCGCTCTCCTTGTACTC	1 422
IF-pEX2D-CmEgt2-DsRed-F	CAAACACCTTCAAACACGTGATGACCGTTACCAATCAGAC	1 371
IF-pEX2D-CmEgt2-DsRed-R	ATGATATCCTTAAGCACGTGAATCTTATACTCCCCATTGG	1 371
IF-pEX2D-NcEgt2-DsRed-F	CAAACACCTTCAAACACGTGATGGTCGCCACCACCGTC	1 422
IF-pEX2D-NcEgt2-DsRed-R	ATGATATCCTTAAGCACGTGGGCGCTCTCCTTGTACTC	1 422

表3 2个物种Egt1和Egt2理化性质分析

Table 3 Physicochemical property analysis of Egt1 and Egt2 in two species

基因 Gene	序列一致性 Sequence identity （%）	蛋白质长度 Protein length （aa）	蛋白质分子量 Molecular weight （kD）	等电点 Isoelectric point （pI）	总平均亲水系数 Grand average of hydropathicity （GRAVY）	信号肽 Signal peptide	亚细胞定位预测 Subcellular localization prediction
NcEgt1	100.00	876	99.02	5.52	-0.460	无No	细胞质Cytoplasm
NcEgt2	100.00	473	53.01	5.55	-0.121	无No	细胞质Cytoplasm
CmEgt1	57.10	890	99.40	5.81	-0.395	无No	细胞质Cytoplasm
CmEgt2	48.78	456	51.66	5.83	-0.235	无No	细胞质Cytoplasm

图2 不同物种Egt1和Egt2的系统发育树（A）及结构域（B）分析A：蛹虫草、白僵菌、冬虫夏草、麦角菌、粗糙脉孢菌、米曲霉、美味牛肝菌、灵芝、灰树花和裂殖酵母的Egt1和Egt2系统进化树分析；B：蛹虫草和粗糙脉孢菌的Egt1和Egt2主要蛋白质结构域

Fig. 2 Phylogenetic tree (A) and domains (B) analysis of Egt1 and Egt2 in different speciesA: Phylogenetic tree analysis of Egt1 and Egt2 in Cordyceps militaris, Beauveria bassiana, Cordyceps sinensis, Claviceps purpurea, Neurospora crassa, Aspergillus oryzae, Boletus edulis, Ganoderma sinense, Grifola frondosa and Schizosaccharomyces pombe. B: The main protein domains of Egt1 and Egt2 in C. militaris and N. crassa

图3 蛹虫草过表达菌株的表型及荧光观察A：将蛹虫草双营养缺陷型菌株CmΔpyrGΔhisB、蛹虫草转化子CmEgt1/CmΔpyrGΔhisB、CmEgt2/CmΔpyrGΔhisB、CmEgt1-CmEgt2/CmΔpyrGΔhisB、NcEgt1/CmΔpyrGΔhisB、NcEgt2/CmΔpyrGΔhisB和NcEgt1-NcEgt2/CmΔpyrGΔhisB在CD、CD+Uri/Ura、CD+His和CD+Uri/Ura+His四种培养基上22 ℃培养7 d的表型；B：从上到下依次是用20倍物镜观察蛹虫草过表达菌株CmEgt1/CmΔpyrGΔhisB、CmEgt2/CmΔpyrGΔhisB、NcEgt1/CmΔpyrGΔhisB、NcEgt2/CmΔpyrGΔhisB、CmEgt1-CmEgt2/CmΔpyrGΔhisB、NcEgt1-NcEgt2/CmΔpyrGΔhisB的荧光图，从左到右分别为GFP荧光、DsRed荧光、微分干涉（DIC），以及DIC、DsRed和GFP合并图像，比例尺=50 μm

Fig. 3 Phenotypic and fluorescence observation of overexpressed strains of C. militarisA: Dual auxotrophic strain CmΔpyrGΔhisB, transformants CmEgt1/CmΔpyrGΔhisB, CmEgt2/CmΔpyrGΔhisB, CmEgt1-CmEgt2/CmΔpyrGΔhisB, NcEgt1/CmΔpyrGΔhisB, NcEgt2/CmΔpyrGΔhisB, and NcEgt1-NcEgt2/CmΔpyrGΔhisB were phenotyped in CD, CD+Uri/Ura, CD+His, and CD+Uri/Ura+His medium at 22 ℃ for 7 d. B: From top to bottom: Fluorescence images of CmEgt1/CmΔpyrGΔhisB, CmEgt2/CmΔpyrGΔhisB, NcEgt1/CmΔpyrGΔhisB, NcEgt2/CmΔpyrGΔhisB, CmEgt1-CmEgt2/CmΔpyrGΔhisB, NcEgt1-NcEgt2/CmΔpyrGΔhisB under 20-fold microscope. Left to right: GFP fluorescence and DsRed fluorescence, differential interference contrast (DIC), merged images of DIC, DsRed, and GFP. Scale bar=50 μm

图4 CmEgt2、NcEgt1和NcEgt2在蛹虫草中的亚细胞定位A：转化子荧光观察，从上到下依次是对照菌株IF-pEX2D-DsRed、转化子CmEgt1/CmΔpyrGΔhisB、CmEgt2/CmΔpyrGΔhisB、NcEgt1/CmΔpyrGΔhisB和NcEgt2/CmΔpyrGΔhisB在63倍镜下菌丝体的显微镜观察；B：CmEgt2、NcEgt1和NcEgt2在蛹虫草中的亚细胞定位，从上到下依次是对照菌株IF-pEX1-GFP、转化子CmEgt2/CmΔpyrGΔhisB与IF-pEX1/CmΔpyrGΔhisB共定位，NcEgt1/CmΔpyrGΔhisB与IF-pEX1/CmΔpyrGΔhisB共定位，以及NcEgt2/CmΔpyrGΔhisB与IF-pEX1/CmΔpyrGΔhisB共定位在63倍镜下菌丝体的显微镜观察，从左至右：GFP荧光、DsRed荧光、DIC，以及DIC、DsRed和GFP合并图像，比例尺=10 μm

Fig. 4 Subcellular localization of CmEgt2, NcEgt1 and NcEgt2 in C. militarisA: Fluorescence observation of transformants, from top to bottom: fluorescence microscopy images of mycelia from the control strain IF‑pEX2D‑DsRed and the transformants CmEgt1/CmΔpyrGΔhisB, CmEgt2/CmΔpyrGΔhisB, NcEgt1/CmΔpyrGΔhisB, and NcEgt2/CmΔpyrGΔhisB under 63-fold microscope. B: Subcellular localization of CmEgt2, NcEgt1, and NcEgt2 in C. militaris, from top to bottom: the mycelium of the control strain IF-pEX1-GFP, co‑localization of transformant CmEgt2/CmΔpyrGΔhisB with IF-pEX1/CmΔpyrGΔhisB, co‑localization of transformant NcEgt1/CmΔpyrGΔhisB with IF-pEX1/CmΔpyrGΔhisB, and co‑localization of transformant NcEgt2/CmΔpyrGΔhisB with IF-pEX1/CmΔpyrGΔhisB under 63-fold microscope. Left to right: GFP fluorescence; DsRed fluorescence; DIC; DIC, DsRed, and GFP merged image, scale bar=10 μm

图5 不同碳源对麦角硫因含量的影响A：蛹虫草野生型菌株在不同碳源培养下麦角硫因的产量；B：蛹虫草野生型菌株在不同碳源培养下麦角硫因的液相色谱峰。DW：干重；EGT：麦角硫因。显著性差异分析采用one-way ANOVA （*P<0.05， ** P<0.01， *** P<0.001），下同

Fig. 5 Effect of different carbon sources on ergothioneine productionA: The ergothioneine production of the wild-type strain of C. militaris in different carbon sources. B: The chromatogram peaks of ergothioneine in the wild-type strain of C. militaris. DW: Dry weight; EGT: ergothioneine. Statistical analyses were performed using one-way ANOVA (*P<0.05, ** P<0.01, *** P<0.001). The same below

图6 不同前体对麦角硫因含量的影响A：蛹虫草野生型菌株在DPY培养基（CK），以及分别添加0.1%的组氨酸、甲硫氨酸、半胱氨酸的麦角硫因含量；B：不同浓度甲硫氨酸处理下麦角硫因的含量

Fig. 6 Effects of different precursors on ergothioneine contentA: Ergothioneine contents of the wild-type strain of C. militaris in DPY medium (CK) and with 0.1% histidine, methionine, and cysteine in the medium, respectively. B: Ergothioneine content under different concentrations of methionine treatment

图7 蛹虫草过表达菌株麦角硫因含量A：蛹虫草野生型和转化子CmEgt1/CmΔpyrGΔhisB、CmEgt1-CmEgt2/CmΔpyrGΔhisB、NcEgt1/CmΔpyrGΔhisB、NcEgt1-NcEgt2/CmΔpyrGΔhisB中麦角硫因含量；B：蛹虫草中麦角硫因的液相色谱峰；C：蛹虫草野生型和转化子NcEgt1-NcEgt2/CmΔpyrGΔhisB在DPY培养基以及添加0.2%甲硫氨酸培养基中的麦角硫因含量；D：蛹虫草CmΔpyrGΔhisB、转化子Cmegt1/CmΔpyrGΔhisB， CmEgt1-CmEgt2/CmΔpyrGΔhisB， Ncegt1/CmΔpyrGΔhisB和NcEgt1-NcEgt2/CmΔpyrGΔhisB在22 ℃使用DPY+Uri/Ura+His培养7 d的表型

Fig. 7 Ergothioneine content of overexpressed strains in C. militarisA: Ergothioneine content in the wild-type and transformants CmEgt1/CmΔpyrGΔhisB, CmEgt1-CmEgt2/CmΔpyrGΔhisB, NcEgt1/CmΔpyrGΔhisB and NcEgt1-NcEgt2/CmΔpyrGΔhisB. B: Chromatogram peaks of ergothioneine in C. militaris. C: Ergothioneine content of the wild-type and transformants NcEgt1-NcEgt2/CmΔpyrGΔhisB in DPY medium and with 0.2% Met in the medium, respectively. D: Phenotypes of CmΔpyrGΔhisB, Cmegt1/CmΔpyrGΔhisB, CmEgt1-CmEgt2/CmΔpyrGΔhisB, Ncegt1/CmΔpyrGΔhisB, NcEgt1-NcEgt2/CmΔpyrGΔhisB cultured on DPY+Uri/Ura + His medium at 22 ℃ for 7 days

图8 蛹虫草过表达菌株虫草素含量A：蛹虫草野生型和转化子CmEgt1/CmΔpyrGΔhisB、CmEgt1-CmEgt2/CmΔpyrGΔhisB、NcEgt1/CmΔpyrGΔhisB、NcEgt1-NcEgt2/CmΔpyrGΔhisB中虫草素含量；B：蛹虫草中虫草素的液相色谱峰。COR：虫草素

Fig. 8 Cordycepin content of overexpressed strains in C. militarisA: Cordycepin content in the wild-type and transformants CmEgt1/CmΔpyrGΔhisB, CmEgt1-CmEgt2/CmΔpyrGΔhisB, NcEgt1/CmΔpyrGΔhisB, NcEgt1-NcEgt2/CmΔpyrGΔhisB. B: Chromatogram peaks of cordycepin in C. militaris. COR: Cordycepin

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