Abstract
Huperzine A (Hup A) is an important drug for treating Alzheimer’s disease (AD) and mainly extracted from the Huperzia serrata (Thunb.) Trevis. (Lycopodiaceae) (HS). Nevertheless, the content of Hup A in HS is very low of 0.007% with growing circle of 8 to 10 years, and the chemical synthesis of Hup A still has some insurmountable limitations in the industrialized production. So, the available resources of Hup A for clinical treatment of AD are scarce. The purpose of this work was to construct a biosynthesis platform based on the endophytic fungi from HS. In this work, five endophytic fungi Mucor racemosus NSH-D, Mucor fragilis NSY-1, Fusarium verticillioides NSH-5, Fusarium oxysporum NSG-1, and Trichoderma harzianum NSW-V were firstly found and isolated from the Chinese folk medicine HS, which were identified according to their morphological characteristics and nuclear ribosomal DNA ITS sequences. The highest efficient fungus could effectively biosynthesize Hup A in a liquid culture of 319.8 ± 0.17 mg/L which were 112 times higher than that of other reported conventional endophytic fungi. Moreover, these fungi with higher hereditary stability could possess the initial expressing ability of Hup A after 40 generations, and the expressed Hup A from these biosynthesis systems has prior physicochemical properties, a better inhibition activity of acetylcholinesterase and a lower cytotoxicity compared with the listed active pharmaceutical ingredients (APIs) of Hup A. These results provide promising alternative resources for producing Hup A at an industrial scale by biosynthesis, and it may also shed light on millions of AD patients.
Key points
• Five novel endophytic fungi with high stability could highly express prior Hup A
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The datasets generated and/or analyzed during the current study are available in the [NCBI] repository, and data generated or analyzed during this study are included in this published article.
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Acknowledgments
The authors would like to express their sincere thankfulness to the Shaanxi Provincial Department of Science and Technology and Education Department, for providing experiment funds, which significantly contribute to the finish of this work. The authors are also greatly thankful to Dr. Zhou Yong-qiang in Nanjing Leyao Venture Capital Management Co. Ltd and Editor Zheng Li-sheng in Chinese Traditional and Herbal Drugs, for the assistance in the AD treatment applications.
Funding
This research work was sponsored by the Youth Innovation Team of Shaanxi Universities (Shaanxi Education No. 90 [2019]), the general programs of Shaanxi Education Department (Grant No.17JS120), the project association for Science and Technology Youth Talent Lift in Colleges and Universities of Shaanxi Province (Grant No.20170411), the scientific research topics of Shaanxi Administration of Traditional Chinese Medicine (Grant no. 2019-ZZ-ZY010), the general programs of Shaanxi Science and Technology department (Grant No. 2020JM-610), field project from Xi’an Medical University (Grant no. 2018PT71, 2018PT72, 2018PT73), and the Science and Technology Innovation Base-Open and Sharing Platform of Science and Technology Resources Project of Shaanxi Province (2019PT-26).
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HWX was responsible for the design of the whole experiment and carried out the morphological and molecular biology studies and drafted the manuscript. HZW participated in the collection of the Huperzia serrata. JM and ZH carried out the preparation of extracts and the test of TLC and HPLC. LWZ was responsible for guiding the research and checking the article. YLB and LF carried out the AChE inhibition ability and cytotoxicity test. HL carried out the test of LC-MS/MS and the study of hereditary stability. ZN participated in the coordination and helped to draft the manuscript. LXF carried out the alkaloid precipitation and acid dye colorimetry and the study of hereditary stability. All authors read and approved the final manuscript.
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The study protocol was approved by the ethics review board of Xi’an Medical University. All of the procedures were performed in accordance with the Declaration of Helsinki and relevant policies in China. The collection of the Huperzia serrata complied with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora and relevant policies in China. All the experiments in the study were carried out in compliance with relevant institutional biosafety and biosecurity protocols and any national or international recommendations relevant to the research field.
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Wen-Xia, H., Zhong-Wen, H., Min, J. et al. Five novel and highly efficient endophytic fungi isolated from Huperzia serrata expressing huperzine A for the treatment of Alzheimer’s disease. Appl Microbiol Biotechnol 104, 9159–9177 (2020). https://doi.org/10.1007/s00253-020-10894-4
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DOI: https://doi.org/10.1007/s00253-020-10894-4