| 摘要: |
| 本论文以蜜环菌属（Armillaria mellea）真菌CPCC 400891为研究对象，基因组挖掘分析发现该菌株基因组中含有18个倍半萜合成酶编码基因，但其功能却鲜有报道。采用色氨酸营养缺陷型表达载体pYET，并以酿酒酵母（Saccharomyces cerevisiae）BJ5464为宿主菌分别表达蜜环菌来源的18个倍半萜合成酶编码基因（Arm_STS1-Arm_STS16）。结合气相色谱-质谱联用仪（gas chromatography-mass spectrometer，GC-MS）分析平台，对构建的重组菌（BJ-Arm_STS1-BJ-Arm_STS16）发酵产物进行采集分析。结果显示，蜜环菌CPCC 400891来源的12个倍半萜合成酶归属于新的类群：亚家族Ⅳ。同时，10个倍半萜合成酶在酿酒酵母中确定表达。根据美国国家标准与技术研究院（NIST）的标准参考数据库（https：//webbook.nist.gov/）检索比对，共鉴定17个倍半萜（醇）分子，包括α-雪松烯、β-雪松烯、罗汉柏烯、杜松萜烯、β-花柏烯和毕橙茄醇等。GC-MS检测分析发现，亚家族Ⅳ类群倍半萜合成酶均能合成多个（种）倍半萜分子。综上所述，本研究为后续改造并优化特定倍半萜合成酶，从而为深入开展STSs的催化机制研究奠定基础，也为从蘑菇类大型真菌中发现更多新颖的倍半萜合成酶提供依据和思考。 |
| 关键词: 蜜环菌属 基因组挖掘 倍半萜合成酶 异源表达 分子系统进化树 功能鉴定 |
| DOI：10.14188/j.ajsh.2023.04.007 |
| 分类号:Q-93 |
| 基金项目:国家自然科学基金（31872617）；中国医学科学院医学与健康科技创新工程（2021-I2M-1-055，2019-I2M-1-005）；北京协和医学院基本科研业务费（3332018097） |
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| Diversity and functional research of sesquiterpene synthases in fungus Armillaria mellea CPCC 400891 |
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LI Yan1,2, FENG Jianju1, LI Jun1, LI Xinxin1, WANG Hao1, YU Liyan1, LI Jian2, ZHANG Tao1
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1.Institute of Medicinal Biotechnology， Chinese Academy of Medical Sciences & Peking Union Medical College， Beijing 100050， China;2.School of Food and Health， Beijing Technology and Business University， Beijing 100048， China
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| Abstract: |
| Genome mining revealed that the genome of Armillaria mellea CPCC 400891 includes eighteen sesquiterpene synthases （STSs） encoding genes， but their functions have been rarely reported. Herein， the eighteen STSs-encoding biosynthetic genes （Arm_STS1-Arm_STS16） were expressed respectively in heterologous host Saccharomyces cerevisiae BJ5464， in which the tryptophan auxotrophic vector pYET served as an expressing plasmid. Eighteen genetically engineered yeast strains （BJ-Arm_STS1-BJ-Arm_STS16） were investigated on gas chromatography-mass spectrometer （GC-MS） analytic platform. Among eighteen sesquiterpene biosynthetic genes recovered from the genome， twelve derived STSs were categorized into new subfamily diverse clade Ⅳ. Moreover， among eighteen STSs coding genes， ten were successfully expressed in yeast. Based on mass spectrometry analysis with NIST chemistry webbook standard reference database （https：//webbook.nist.gov/）， seventeen sesquiterpenes and sesquiterpene-derived alcohols including α-himachalene， β-himachalene， cis-thujopsene， β-cadinene， β-chamigrene， and β-cadinol were identified. Additionally， GC-MS analysis revealed that the STSs of group clade Ⅳ could produce several sesquiterpenes. Our report could lay foundation for further optimizing specific sesquiterpene synthases and investigating catalytic mechanism of STSs. This study also provides reference and understandings for mining more new STSs from large fungi mushrooms. |
| Key words: Armillaria genome mining sesquiterpene synthase heterologous expression phylogenetic tree functional identification |
