金耳和毛韧革菌麦角硫因生物合成基因的克隆及生物信息学分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0116

生物技术通报 ›› 2024, Vol. 40 ›› Issue (7): 259-272.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0116

金耳和毛韧革菌麦角硫因生物合成基因的克隆及生物信息学分析

沈真辉¹^,²^,³^,⁴(), 曹瑶¹^,²^,³^,⁴, 杨林雷¹^,²^,³^,⁴, 罗祥英¹^,²^,³^,⁴, 子灵山¹^,², 陆青青¹^,², 李荣春¹^,²^,³^,⁴()

1.云南菌视界生物科技有限公司，昆明 650200
2.云南省级（珍稀食用菌）企业工程技术中心，昆明 650200
3.云南省张劲松专家工作站，昆明 650200
4.云南（昆明）张劲松药用真菌专家工作站，昆明 650200

收稿日期:2024-01-31 出版日期:2024-07-26 发布日期:2024-07-30
通讯作者: 李荣春，教授，硕士生导师，研究方向：食用菌栽培与资源评价；E-mail: rongchunli@126.com
作者简介:沈真辉，硕士，中级农艺师，研究方向：食用菌基础分子生物学；E-mail: 1947843434@qq.com
基金资助:
云南省张劲松专家工作站(202305AF150084);云南昆明张劲松药用真菌专家工作站(YSZJGZZ-2022043)

Cloning and Bioinformatics Analysis of the Ergothioneine Biosynthesis Genes in Naematelia aurantialba and Stereum hirsutum

SHEN Zhen-hui¹^,²^,³^,⁴(), CAO Yao¹^,²^,³^,⁴, YANG Lin-lei¹^,²^,³^,⁴, LUO Xiang-ying¹^,²^,³^,⁴, ZI Ling-shan¹^,², LU Qing-qing¹^,², LI Rong-chun¹^,²^,³^,⁴()

1. Yunnan Junshijie Biotechnology Ltd., Kunming 650200
2. Yunnan Provincial（Rare Edible Mushroom）Enterprise Engineering Technology Centre, Kunming 650200
3. Yunnan Province Zhang Jinsong Expert Workstation, Kunming 650200
4. Yunnan（Kunming）Zhang Jinsong Medicinal Fungus Expert Workstation, Kunming 650200

Received:2024-01-31 Published:2024-07-26 Online:2024-07-30

摘要/Abstract

摘要：

【目的】 探究金耳和毛韧革菌麦角硫因生物合成途径。【方法】 利用PCR扩增技术分别克隆金耳和毛韧革菌的麦角硫因合成酶基因Egt1和Egt2并利用生物信息学软件分析其功能；高效液相色谱技术鉴定2个物种的组氨酸三甲基内盐中间产物、麦角硫因及其含量。【结果】 成功克隆得到了2个物种Egt1和Egt2基因的完整DNA序列。生物信息学分析表明，2个物种的Egt1均含有EgtD和SAM依赖性甲基转移酶等功能结合域，Egt2均含有磷酸吡哆醛（LPL）和半胱氨酸脱硫酶的结合位点； Egt1和Egt2与裂殖酵母和粗糙脉孢菌等模型真菌具有相似的功能域和底物结合位点，表明Egt1和Egt2可能与这些模式真菌具有相似的基因功能。高效液相色谱法分析表明，金耳芽孢（JEYB）、毛韧革菌发酵液（ShFJY）、菌丝体（ShJST）及金耳子实体（JEZST）中均含有组氨酸三甲基内盐和麦角硫因，并且金耳子实体麦角硫因含量最高（113.19 μg/g），分别是金耳芽孢、毛韧革菌发酵液和毛韧革菌菌丝体的7.45倍、26.14倍和27.74倍。【结论】 首次鉴定了金耳和毛韧革菌的Egt1和Egt2基因。推测金耳和毛韧革菌的生物合成途径都是由组氨酸在Egt1酶催化形成组氨酸三甲基内盐，再由Egt1酶催化形成海西烯半胱氨酸亚砜，最后由Egt2酶催化最终形成麦角硫因。

关键词: 金耳, 毛韧革菌, 麦角硫因, 基因克隆, 生物合成

Abstract:

【Objective】 To explore the biosynthetic pathway of ergothioneine in Naematelia aurantialba and Stereum hirsutum. 【Method】 The ergothioneine synthase gene Egt1 and Egt2 of N. aurantialba and S. hirsutum were cloned by PCR amplification technology, respectively, and their functions were analyzed by bioinformatics software. High performance liquid chromatography（HPLC）was used to identify the intermediate products of hercynine, ergothioneine and their contents in two species. 【Result】 The complete DNA sequences of Egt1 and Egt2 genes of two species were successfully cloned. The analysis using bioinformatics software revealed that Egt1 of the two species contained functional binding domains such as EgtD and SAM-dependent methyltransferase. Egt2 contained the binding sites for pyridoxal phosphate（LPL）and cysteine desulfurase. Further analysis indicated that Egt1 and Egt2 shared similar functional domains and substrate binding sites with model fungi（Schizosaccharomyces pombe and Neurospora crassa）. This result showed that Egt1 and Egt2 may have similar gene functions as these model fungi. HPLC analysis revealed the presence of hercynine and ergothioneine in N. aurantialba blastospore （JEYB）, S. hirsutum fermentum broth（ShFJY）, S. hirsutum mycelium（ShJST）and N. aurantialba fruiting bodies（JEZST）. Additionally, the ergothioneine content in the JEZST was found to be the highest at 113.19 μg/g, which was 7.45 times, 26.14 times, and 27.74 times higher than that of the JEYB, ShFJY, and ShJST, respectively. 【Conclusion】 The Egt1 and Egt2 genes of N. aurantialba and S. hirsutum were identified for the first time. It is hypothesized that the biosynthetic pathway of N. aurantialba and S. hirsutum are involved the catalysis of histidine by the Egt1 enzyme, resulting in the formation of hercynine. Subsequently, the Egt1 enzyme catalyzes the conversion of hercynine into hercynylcysteine sulfoxide. Finally, the Egt2 enzyme catalyzes the transformation of hercynylcysteine sulfoxide into ergothioneine.

Key words: N. aurantialba, S. hirsutum, ergothioneine, gene cloning, biosynthesis

沈真辉, 曹瑶, 杨林雷, 罗祥英, 子灵山, 陆青青, 李荣春. 金耳和毛韧革菌麦角硫因生物合成基因的克隆及生物信息学分析[J]. 生物技术通报, 2024, 40(7): 259-272.

SHEN Zhen-hui, CAO Yao, YANG Lin-lei, LUO Xiang-ying, ZI Ling-shan, LU Qing-qing, LI Rong-chun. Cloning and Bioinformatics Analysis of the Ergothioneine Biosynthesis Genes in Naematelia aurantialba and Stereum hirsutum[J]. Biotechnology Bulletin, 2024, 40(7): 259-272.

图/表 12

图1 微生物麦角硫因生物合成途径 SAM，SAH（或AdoHcy），Cys和PLP分别表示s-腺苷甲硫氨酸，S腺苷同型半胱氨酸，半胱氨酸和磷酸吡哆醛

Fig. 1 Biosynthetic pathways of ergothioneine in microorganism SAM, SAH (or AdoHcy), Cys and PLP refers to s-adenosylmethionine, S-adenosylhomocysteine, cysteine and pyridoxal phosphate respectively

图2 不同样品形态特征 A：金耳芽孢（JEYB）；B：毛韧革菌液体菌种（ShFJY+ShJST）；C：金耳子实体（JEZST）

Fig. 2 Morphological characteristics of different samples A: N. aurantialba blastospore （JEYB）；B: S. hirsutum liquid culture（ShFJY+ShJST）；C: N. aurantialba fruiting body（JEZST）

表1 金耳和毛韧革菌麦角硫因生物合成基因引物信息

Table 1 Primer information for ergothioneine biosynthetic genes of N. aurantialba and S. hirsutum

序号Serial No.	引物名称 Primer name	序列 Sequence（5'-3'）	退火温度 Annealing temperature/℃
1	ShEgt1-F	TGCGTCGGTCTCCTTTCTT	53
2	ShEgt1-R	CGGAGCAGAACAGATTTATCG
3	ShEgt2-F	CACCACCACACATCACAGTC	56
4	ShEgt2-R	GACAAGTTCACAACGAGCAT
5	NaEgt1-F	TGTCTGTCTCCTCCATCCA	56
6	NaEgt1-R	TAGTCCCAGGCTCATCACA
7	NaEgt2-F	GTATCGCTGTTTACCACCTGT	56
8	NaEgt2-F	TCTACTCATCCTTCGTCGG

表2 本研究所用的生物信息学软件

Table 2 Bioinformatics software used in this study

序号Serial No.	分析软件Analysis software	用途Application	网址Web site
1	ORF	CDS序列预测CDS sequence prediction	https://www.ncbi.nlm.nih.gov/orffinder
2	Gene Structure Display Server	绘制基因结构图Drawing a genetic structure map	http://gsds.gao-lab.org/
3	NCBI-Blast	基因同源性分析Gene homology analysis	https://blast.ncbi.nlm.nih.gov/Blast.cgi
4	ExPASy- ProtParam	分析蛋白的理化性质Analyzing the physicochemical properties of proteins	https://web.expasy.org/protparam/
5	SignalP4.1	信号肽预测Prediction of signal peptide	http://genome.cbs.dtu.dk/services/SignalP-4.1/
6	PSORT II	亚细胞定位Subcellular localization	https://www.genscript.com/psort.html
7	CCD	保守结构域预测Conservative structural domain prediction	http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
8	PSIPRED	二级结构预测Secondary structure of protein prediction	https://www.novopro.cn/tools/secondary-structure-prediction.html
9	SWISS-MODEL	三级结构预测Tertiary structure prediction	https://swissmodel.expasy.org/
10	Phyre2	三级结构配体预测Tertiary structure ligand prediction	http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index
11	MEGA7.0	系统进化树分析Phylogenetic analysis	https://www.megasoftware.net/

图3 金耳和毛韧革菌的Egt1和Egt2基因PCR扩增验证（A，B）及基因结构分析（C） M：DL2000 marker；ShJST：毛韧革菌菌丝体；JEYB：金耳芽孢；JEZST：金耳子实体

Fig. 3 Verification of PCR amplification of Egt1 and Egt2 genes of N. aurantialba and S. hirsutum（A, B）and gene structure analysis（C） M: DL2000 marker; ShJST: S. hirsutum mycelium; JEYB: N. aurantialba blastospore ; JEZST: N. aurantialba fruiting body

表3 两个物种Egt1和Egt2理化性质分析

Table 3 Analysis of physical and chemical properties of Egt1 and Egt2 in two species

基因 Gene	CDS长度CDS length/bp	蛋白质长度 Protein length	蛋白质分子量Molecular weight/kD	等电点 Isoelectric point	总平均亲水系数Grand average of hydropathicity （GRAVY）	信号肽 Signal peptide	细胞中分布 Location
NaEgt1	2952	984	110.32	5.30	-0.479	无No	细胞质Cytoplasmic
ShEgt1	2565	885	99.03	5.19	-0.528	无No	细胞质Cytoplasmic
NaEgt2	2178	726	81.61	5.87	-0.368	无No	细胞质Cytoplasmic
ShEgt2	1383	461	51.46	5.68	-0.238	无No	细胞质Cytoplasmic

图4 五个真菌Egt1和Egt2蛋白三级结构及结合位点分析 A：5个真菌Egt1和Egt2（Egt3）的三级结构比较；B：Egt1与咪唑（红色箭头）和醋酸根离子（蓝色箭头）结合；C：Egt1与s-腺苷甲硫氨酸（SAM）的结合；D：EgtD与Fe3+的结合；E：Egt1与半胱氨酸（红色箭头）结合；F：Egt2与S-巯基半胱氨酸（红色箭头）结合；G-H：Egt2与两个磷酸吡哆醛（PLP）位点结合。SpEgt1、NcEgt1、FvEgt1、SpEgt2、NcEgt2、FvEgt2及FvEgt3分别表示裂殖酵母、粗糙脉孢霉、金针菇的麦角硫因生物合成关键酶（登录号：CAA22334.2、XP_956324.3、QBB19872.1、NP_595091.1、A7UX13、QBB19873.1及QBB19874.1）

Fig. 4 Tertiary structure and binding site analysis of five fungal Egt1 and Egt2 proteins A: Comparison of the tertiary structures of five fungal Egt1 and Egt2（Egt3）; B: Egt1 binds to imidazole（red arrow）and acetate ions（blue arrow）; C: binding of Egt1 to s-adenosylmethionine（SAM）; D: the combination of EgtD and Fe3+; E: Egt1 binds to cysteine（red arrow）; F: Egt2 binds to S-mercaptocysteine; G-H: Egt2 binds to two pyridoxal phosphate（PLP）sites. SpEgt1, NcEgt1, FvEgt1, SpEgt2, NcEgt2, FvEgt2 and FvEgt3 are key enzyme for ergothioneine biosynthesis of S. pombe, N. crassa and F. filiformis, respectively（Accession number: CAA22334.2, XP_956324.3, QBB19872.1, NP_595091.1, A7UX13, QBB19873.1 and QBB19874.1）.

表4 5种真菌的Egt1和Egt2蛋白三级结构预测信息

Table 4 Predicted information on the tertiary structure of Egt1 and Egt2 proteins from five fungal species

蛋白 Protein	物种 Species	模板 Template	覆盖度 Coverage/%	可信度 Confidence/%	功能域一致性 Functional domain identity/%	结合位点 Binding site
Egt1	耻垢分枝杆菌 M smegmatis	c4uy5A	51/46/55/36/73	100/100/100/100/100	34/32/26/24/35	咪唑和醋酸根离子 Imidazole and acetate ion
	大利什曼原虫 Leishmania major	d1xtpa	23/35/38/-/59	96/98/99/-/98	31/30/18/-/19	s-腺苷甲硫氨酸 S-adenosyl-methionine（SAM）
	耐热分枝杆菌 Mycobacterium thermoresistibile	c4x8bA	44/49/55/57/-	100/100/100/100/-	27/26/23/24/-	Fe³⁺离子 Fe³⁺ ion
	嗜热氢单胞菌 Hydrogenimonas thermophila	c8khqD	44/48/55/57/-	100/100/100/100/-	26/26/24/24/-	半胱氨酸 Cysteine（Cys）
Egt2	粗糙脉孢霉 N. crassa	c5utsC	59/91/97/94/93/92	100/100/100/100/100/100	39/35/31/97/21/36	-
Egt2	集胞藻属 Synechocystis sp.	d1elua	56/88/95/90/90/89	99.9/100/100/100/100/100	20/19/16/16/18/16	磷酸吡哆醛和S-巯基半胱氨酸 Pyridoxal phosphate and S-mercaptocysteine

图5 不同物种Egt1（A）和Egt2（B）系统进化树分析紫色标注表示毛韧革菌和金耳的Egt2序列比对结果；红色标注表示毛韧革菌与其他真菌的Egt2序列比对结果；蓝色标注表示金耳与其他真菌的Egt2序列比对结果

Fig. 5 Phylogenetic tree analysis of Egt1（A）and Egt2（B）in different species Purple colours indicate the results of Egt2 sequence comparison between S. hirsutum and N. aurantialba; red colours indicate the results of Egt2 sequence comparison between S. hirsutum and other fungi; blue colours indicate the results of Egt2 sequence comparison between N. aurantialba and other fungi

图6 标准品（A）和金耳子实体（JEZST）样品（B）的HPLC图谱，组氨酸三甲基内盐（C）和麦角硫因（D）标准曲线

Fig 6 Chromatograms of reference substances（A）and N. aurantialba fruiting body（JEZST）（B）, standard curve of hercynine and L-ergothioneine

图7 不同样品组氨酸三甲基内盐和麦角硫因含量测定 A：液体样品；B：固体样品。YTPDA-CK：PDA液体培养基（对照）；ShFJY：毛韧革菌发酵液；ShJST：毛韧革菌菌丝体；JSJ-JEYB：金耳芽孢；JEZST：金耳子实体。*，**和***分别表示在P<0.05、P<0.01和P<0.0001水平上差异显著

Fig. 7 Determination of hercynine and ergothioneine content in different samples A: Liquid sample; B: solid sample. YTPDA-CK: PDA liquid medium（control）; ShFJY: S. hirsutum fermentation broth; ShJST: S. hirsutum mycelium; JSJ-JEYB: N. aurantialba blastospa; JEZST: N. aurantialba fruiting body. *, ** and *** indicate a significant difference at P<0.05, P<0.01 and P<0.0001, respectively

图8 金耳和毛韧革菌假定麦角硫因生物合成途径

Fig. 8 Putative biosynthetic pathway of ergothioneine in N. aurantialba and S. hirsutum

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金耳和毛韧革菌麦角硫因生物合成基因的克隆及生物信息学分析

Cloning and Bioinformatics Analysis of the Ergothioneine Biosynthesis Genes in Naematelia aurantialba and Stereum hirsutum

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