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Isolation of endophytic fungi and screening of Huperzine A-producing fungus from Huperzia serrata in Vietnam.

ABSTRACT: Huperzine A (HupA), a natural Lycopodium alkaloid derived from Huperzia serrata (Thunb. ex Murray) Trev. plants, is a highly active acetylcholinesterase inhibitor and a key compound used for treating Alzheimer's disease (AD). Recently, HupA has been reported in various endophytic fungi isolated from H. serrata. In the present study, 153 endophytic fungi were isolated from healthy tissues of H. serrata collected from natural populations in Lam Dong province of Central Vietnam. The endophytic fungi were identified based on morphological characteristics and Internal Transcribed Spacer sequences. Among them, 34 strains were classified into seven genera belonging to Ascomycota, including Alternaria, Fusarium, Trichoderma, Penicillium, Paecilomyces, and Phoma, and eight strains belonging to the genus Mucor (Zygomycota). The other strains remained unidentified. According to the results of thin-layer chromatography and high-performance liquid chromatography, only one of the 153 strains, Penicillium sp. LDL4.4, could produce HupA, with a yield 1.38?mg?l-1 (168.9?µg?g-1 dried mycelium) when cultured in potato dextrose broth, which was considerably higher than that of other reported endophytic fungi. Such a fungus is a promising candidate and alternative to presently available HupA production techniques for treating AD and preventing further memory decline.

SUBMITTER: Thi Minh Le T

PROVIDER: S-EPMC6834622 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Isolation of endophytic fungi and screening of Huperzine A-producing fungus from Huperzia serrata in Vietnam.

Thi Minh Le Thanh T Thi Hong Hoang Anh A Thi Bich Le Thuy T Thi Bich Vo Thuy T Van Quyen Dong D Hoang Chu Ha H

Scientific reports 20191106 1

Huperzine A (HupA), a natural Lycopodium alkaloid derived from Huperzia serrata (Thunb. ex Murray) Trev. plants, is a highly active acetylcholinesterase inhibitor and a key compound used for treating Alzheimer's disease (AD). Recently, HupA has been reported in various endophytic fungi isolated from H. serrata. In the present study, 153 endophytic fungi were isolated from healthy tissues of H. serrata collected from natural populations in Lam Dong province of Central Vietnam. The endophytic fung ...[more]

PMID: 31695105

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Project description:As the population ages globally, there seem to be more people with Alzheimer's disease. Unfortunately, there is currently no specific treatment for the disease. At present, Huperzine A (HupA) is one of the best drugs used for the treatment of Alzheimer's disease and has been used in clinical trials for several years in China. HupA was first separated from Huperzia serrata, a traditional medicinal herb that is used to cure fever, contusions, strains, hematuria, schizophrenia, and snakebite for several hundreds of years in China, and has been confirmed to have acetylcholinesterase inhibitory activity. With the very slow growth of H. serrata, resources are becoming too scarce to meet the need for clinical treatment. Some endophytic fungal strains that produce HupA were isolated from H. serrate in previous studies. In this article, the diversity of the endophytic fungal community within H. serrata was observed and the relevance to the production of HupA by the host plant was further analyzed. A total of 1167 strains were obtained from the leaves of H. serrata followed by the stems (1045) and roots (824). The richness as well as diversity of endophytic fungi within the leaf and stem were higher than in the root. The endophytic fungal community was similar within stems as well as in leaves at all taxonomic levels. The 11 genera (Derxomyces, Lophiostoma, Cyphellophora, Devriesia, Serendipita, Kurtzmanomyces, Mycosphaerella, Conoideocrella, Brevicellicium, Piskurozyma, and Trichomerium) were positively correlated with HupA content. The correlation index of Derxomyces with HupA contents displayed the highest value (CI = 0.92), whereas Trichomerium showed the lowest value (CI = 0.02). Through electrospray ionization mass spectrometry (ESI-MS), it was confirmed that the HS7-1 strain could produce HupA and the total alkaloid concentration was 3.7 ug/g. This study will enable us to screen and isolate the strain that can produce HupA and to figure out the correlation between endophytic fungal diversity with HupA content in different plant organs. This can provide new insights into the screening of strains that can produce HupA more effectively.

Project description:Plants maintain the steady-state balance of the mutually beneficial symbiosis relationship with their endophytic fungi through secondary metabolites. Meanwhile endophytic fungi can serve as biological inducers to promote the biosynthesis and accumulation of valuable secondary metabolites in host plants through a variety of ways. The composition and structure of endophytic fungal community are affected by many factors, including tissues, seasons and so on. In this work, we studied the community diversity, temporal and spatial pattern of endophytic fungi detected from the roots, stems and leaves of Huperzia serrata in different seasons. The correlation between endophytic fungi and huperzine A (HupA) content in plants was analyzed. A total of 7005 operational taxonomic units were detected, and all strains were identified as 14 phyla, 54 classes, 140 orders, 351 families and 742 genera. Alpha diversity analysis showed that the diversity of endophytic fungi in stem and leaf was higher than that in root, and the diversity in summer (August) was lower than that in other months. NMDS analysis showed that the endophytic fungal communities of leaves, stems and roots were significantly different, and the root and leaf communities were also different between four seasons. Through correlation analysis, it was found that 33 genera of the endophytic fungi of H. serrata showed a significant positive correlation with the content of HupA (p < 0.05), of which 13 genera (Strelitziana, Devriesia, Articulospora, Derxomyces, Cyphellophora, Trechispora, Kurtzmanomyces, Capnobotryella, Erythrobasidium, Camptophora, Stagonospora, Lachnum, Golubevia) showed a highly significant positive correlation with the content of HupA (p < 0.01). These endophytic fungi may have the potential to promote the biosynthesis and accumulation of HupA in plant. This report is the first time to analyze the diversity of endophytic fungi in tissues of H. serrata in different seasons, which proves that there is variability in different tissues and seasonal distribution patterns. These findings provide references to the study of endophytic fungi of H. serrata.

Project description:BackgroundHuperzia serrata (H. serrata) is an economically important traditional Chinese herb with the notably medicinal value. As a representative member of the Lycopodiaceae family, the H. serrata produces various types of effectively bioactive lycopodium alkaloids, especially the huperzine A (HupA) which is a promising drug for Alzheimer's disease. Despite their medicinal importance, the public genomic and transcriptomic resources are very limited and the biosynthesis of HupA is largely unknown. Previous studies on comparison of 454-ESTs from H. serrata and Phlegmariurus carinatus predicted putative genes involved in lycopodium alkaloid biosynthesis, such as lysine decarboxylase like (LDC-like) protein and some CYP450s. However, these gene annotations were not carried out with further biochemical characterizations. To understand the biosynthesis of HupA and its regulation in H. serrata, a global transcriptome analysis on H. Serrata tissues was performed.ResultsIn this study, we used the Illumina Highseq4000 platform to generate a substantial RNA sequencing dataset of H. serrata. A total of 40.1 Gb clean data was generated from four different tissues: root, stem, leaf, and sporangia and assembled into 181,141 unigenes. The total length, average length, N50 and GC content of unigenes were 219,520,611 bp, 1,211 bp, 2,488 bp and 42.51%, respectively. Among them, 105,516 unigenes (58.25%) were annotated by seven public databases (NR, NT, Swiss-Prot, KEGG, COG, Interpro, GO), and 54 GO terms and 3,391 transcription factors (TFs) were functionally classified, respectively. KEGG pathway analysis revealed that 72,230 unigenes were classified into 21 functional pathways. Three types of candidate enzymes, LDC, CAO and PKS, responsible for the biosynthesis of precursors of HupA were all identified in the transcripts. Four hundred and fifty-seven CYP450 genes in H. serrata were also analyzed and compared with tissue-specific gene expression. Moreover, two key classes of CYP450 genes BBE and SLS, with 23 members in total, for modification of the lycopodium alkaloid scaffold in the late two stages of biosynthesis of HupA were further evaluated.ConclusionThis study is the first report of global transcriptome analysis on all tissues of H. serrata, and critical genes involved in the biosynthesis of precursors and scaffold modifications of HupA were discovered and predicted. The transcriptome data from this work not only could provide an important resource for further investigating on metabolic pathways in H. serrata, but also shed light on synthetic biology study of HupA.

Dataset Information

Isolation of endophytic fungi and screening of Huperzine A-producing fungus from Huperzia serrata in Vietnam.

Publications

Isolation of endophytic fungi and screening of Huperzine A-producing fungus from Huperzia serrata in Vietnam.

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