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

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(Bacillus amyloliquefaciens)，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。

Abstract:

[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 with strain LP22 for 48 h increased the content of taxanes and flavonoids in T. media. The findings provide a basis for further research on the fermentation and processing of T. media.