Aims: To characterize and identify a novel Huperzine A (HupA)‐producing fungal strain Slf14 isolated from Huperzia serrata (Thunb. ex Murray) Trev. in China. Methods and Results: The isolation, identification and characterization of a novel endophytic fungus producing HupA specifically and consistently from the leaves of H. serrata were investigated. The fungus was identified as Shiraia sp. Slf14 by molecular and morphological methods. The HupA produced by this endophytic fungus was shown to be identical to authentic HupA analysed by thin layer chromatographic, High‐performance liquid chromatography (HPLC), LC‐MS, ¹H NMR and acetylcholinesterase (AChE) inhibition activity in vitro. The amount of HupA produced by Shiraia sp. Slf14 was quantified to be 327·8 μg l⁻¹ by HPLC, which was far higher than that of the reported endophytic fungi, Acremonium sp., Blastomyces sp. and Botrytis sp. Conclusions: The production of HupA by endophyte Shiraia sp. Slf14 is an enigmatic observation. It would be interesting to further study the HupA production and regulation by the cultured endophyte in H. serrata and in axenic cultures. Significance and Impact of the Study: Although the current accumulation of HupA by the endophyte is not very high, it could provide a promising alterative approach for large‐scale production of HupA. However, further strain improvement and the fermentation process optimization are required to result in the consistent and dependable production.

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.

Here, we report the draft genome sequence of Shiraia sp. strain Slf14 (China Center for Type Culture Collection [CCTCC] no. 209294), which is used to produce huperzine A and hypocrellin A. The genome sequence will allow for the characterization of the molecular mechanisms underlying its beneficial properties.

This study attempted to improve the polyphenol and volatile aroma compound contents in Nanfeng tangerine wines using non-Saccharomyces yeast and Saccharomyces cerevisiae. The effects of fermentation with pure cultures of Candida ethanolica, Hanseniaspora guilliermondii and Hanseniaspora thailandica, as well as in sequential and mixed inoculations (1:1 or 1:100 ratio) with S. cerevisiae in Nanfeng tangerine wines were evaluated. C. ethanolica was found to produce the most polyphenols (138.78 mg/L) during pure fermentation, while H. guilliermondii produced the most volatile aroma compounds (442.34 mg/L). The polyphenol content produced during sequential fermentation with S. cerevisiae and H. guilliermondii (140.24 mg/L) or C. ethanolica (140.21 mg/L) was significantly higher than other co-fermentations. Meanwhile, the volatile aroma compounds were found to be more abundant in S. cerevisiae/H. guilliermondii mixed fermentation (1:1 ratio) (588.35 mg/L) or S. cerevisiae/H. guilliermondii sequential fermentation (549.31 mg/L). Thus, S. cerevisiae/H. guilliermondii sequential fermentation could considerably boost the polyphenol and volatile aroma component contents in Nanfeng tangerine wines. The findings of this study can be used to drive strategies to increase the polyphenol content and sensory quality of tangerine wines and provide a reference for selecting the co-fermentation styles for non-Saccharomyces yeast and S. cerevisiae in fruit wine fermentation.