Isolation and characterization of endophytic huperzine A-producing fungi from Huperzia serrata

Huperzia serrata is a producer of huperzine A (HupA), a cholinesterase inhibitor (ChEI). Over 120 endophytic fungi were recovered from this plant and screened for Hup-A and nine were found. These nine represented seven different fungal genera with the most significant producer being Shiraia sp. A total of 127 endophytic fungi isolates obtained from the root, stem, and leaf segments of H. serrata were grouped into 19 genera based on their morphological traits and sequence analysis of the internal transcribed spacers (ITS1-5.8S-ITS2), indicating endophytic fungi in H. serrata are diverse and abundant. Aspergillus, Podospora, Penicillium, Colletotrichum, and Acremonium were the frequent genera, whereas the remaining genera were infrequent groups. Overall, 39 endophytic fungi isolates showed acetylcholinesterase (AChE) inhibition in vitro. Nine endophytic fungi isolates from seven distinct genera were capable of producing HupA verified by thin-layer chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Among the HupA-producing fungi, the yield of HupA produced by the Shiraia sp. Slf14 was 327.8 μg/l in potato dextrose broth, and the fungal HupA was further validated by mass spectrometry (ESI-MS). The present study demonstrated that H. serrata was a fascinating fungal reservoir for producing HupA and other ChEIs.