高产紫杉烷内生菌的分离筛选及曼地亚红豆杉发酵炮制  

Endophytic bacteria and fungi for high production of taxanes:screening and application in the fermentation of Taxus media

作　　者：郭幸 王胜超[1] 张振凌[1,2,3,4,5] 李雅静 吴亚宁 张帅 院军 GUO Xing;WANG Shengchao;ZHANG Zhenling;LI Yajing;WU Yaning;ZHANG Shuai;YUAN Jun(School of Pharmacy,Henan University of Chinese Medicine,Zhengzhou 450046,Henan,China;Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases Co-constructed by Henan province&Education Ministry of China,Zhengzhou 450046,Henan,China;Engineering Research Center of Traditional Chinese Medicine Processing Technology of Henan Province,Zhengzhou 450046,Henan,China;Key Laboratory of Chinese Medicine for Preparation of TCM Decoction Pieces of Henan Province,Zhengzhou 450046,Henan,China;Collaborative Innovation Center of Research and Development on the Whole Industry Chain of Yu-Yao of Henan Province,Zhengzhou 450046,Henan,China)

机构地区：[1]河南中医药大学药学院,河南郑州450046 [2]呼吸疾病中医药防治省部共建协同创新中心,河南郑州450046 [3]河南省中药特色炮制技术工程研究中心,河南郑州450046 [4]河南省中药饮片炮制中医药重点实验室,河南郑州450046 [5]豫药全产业链研发河南省协同创新中心,河南郑州450046

出　　处：《微生物学通报》2024年第7期2647-2662,共16页Microbiology China

基　　金：2023年郑州市协同创新专项(2023XTCX041);河南中医药大学2022年度研究生科研创新项目(2022KYCX032)。

摘　　要：【背景】曼地亚红豆杉内生菌种类丰富,能产生多种抗肿瘤活性成分;经过中药发酵炮制,部分有效成分的含量得以提高。【目的】选用曼地亚红豆杉产紫杉烷的内生菌作为发酵菌种,优化曼地亚红豆杉固体发酵的优势菌株及发酵时间,创新红豆杉炮制方法。【方法】采用平板划线法对曼地亚红豆杉内生菌进行分离纯化,通过HPLC及UPLC-MS测定紫杉烷类化合物,以筛选高产紫杉烷成分的内生菌。筛选出的菌株用于发酵炮制曼地亚红豆杉,采用HPLC法测定7种成分含量,结合权重计算得分,优选曼地亚红豆杉固体发酵炮制的优势菌株及时间,并通过16S rRNA基因及全基因组测序对优势菌株进行分类鉴定。【结果】从曼地亚红豆杉中分离出30株真菌、35株细菌,筛选出7株内生菌产紫杉烷;其中6株产巴卡亭Ⅲ、4株产三尖杉宁碱、2株产紫杉醇,6株产巴卡亭Ⅲ的内生菌产量分别为LY2(2.99 mg/L)、LY9(4.90 mg/L)、LP22(0.34 mg/L)、PJ20(1.43 mg/L)、PJ25(0.18 mg/L)、PJ30(0.36 mg/L),4株产三尖杉宁碱的内生菌产量分别为:LY2(4.92 mg/L)、LY9(1.31 mg/L)、PP4(0.66 mg/L)、PJ25(1.31 mg/L),2株产紫杉醇的内生菌产量分别为:PP4(0.70 mg/L)、PJ25(0.34 mg/L)。HPLC含量测定及权重计算得分显示,菌株LP22为曼地亚红豆杉固体发酵炮制的优势菌株,最佳发酵时间为48 h,优势菌株LP22经16S rRNA基因及全基因组学测序鉴定为解淀粉芽孢杆菌(Bacillusamyloliquefaciens),基因组测序数据提交至NCBI数据库获得登录号为PRJNA1039587。【结论】曼地亚红豆杉部分内生菌能够产紫杉烷,将菌株LP22加入药粉固体发酵48 h,有利于提高曼地亚红豆杉中的紫杉烷及黄酮含量,为后续曼地亚红豆杉发酵炮制研究奠定了基础。[Background]Taxus media are abundant in endophytic bacteria and fungi,which can produce a range of anti-tumor active compounds.Fermentation and processing can increase the concentrations of active components in Chinese herbal medicines.[Objective]To select the optimal strain from the taxane-producing endophytic strains of T.media for solid fermentation and optimize the fermentation duration,so as to innovate the solid fermentation method of T.media.[Methods]The plate streaking method was used to isolate the endophytic strains of T.media.HPLC and UPLC-MS were employed to measure taxanes,and the strains with high yields of taxanes were selected.The selected strains were used for solid fermentation of T.media,and the concentrations of seven components were determined by HPLC.Weighted scores were computed to determine the optimal strain for fermentation,and the optimal duration of the solid fermentation process was determined.The optimal strain was identified by 16S rRNA gene and whole-genome sequencing.[Results]A total of 30 fungal strains and 35 bacterial strains were isolated from T.media,and 7 strains had the ability to produce taxanes.Among them,6,4,and 2 strains produced baccatin Ⅲ,cephalomannine,and taxol,respectively.The 6 strains producing baccatin Ⅲ were LY2(2.99 mg/L),LY9(4.90 mg/L),LP22(0.34 mg/L),PJ20(1.43 mg/L),PJ25(0.18 mg/L),and PJ30(0.36 mg/L).The 4 strains yielding cephalomannine were LY2(4.92 mg/L),LY9(1.31 mg/L),PP4(0.66 mg/L),and PJ25(1.31 mg/L).The 2 strains capable of producing taxol were PP4(0.70 mg/L)and PJ25(0.34 mg/L).The HPLC results and weighted scores confirmed that LP22 was the predominant strain for solid fermentation and 48 h was the optimum fermentation period.The strain LP22 was identified as Bacillus amyloliquefaciens by 16S rRNA gene and whole genome sequencing.The full-length genome sequencing data was submitted to NCBI and received the accession number PRJNA1039587.[Conclusion]Certain endophytic strains of T.media have the ability to produce taxanes.The solid fermentation wi

关 键 词：曼地亚红豆杉 内生菌 紫杉醇 发酵 

分 类 号：TQ920.1[轻工技术与工程—发酵工程] TQ461