Jin et al. AMB Expr (2020) 10:105 https://doi.org/10.1186/s13568-020-01039-x ORIGINAL ARTICLE Open Access Genome sequencing and analysis of fungus Hirsutella sinensis isolated from Ophiocordyceps sinensis Li‑Qun Jin1, Zhe‑Wen Xu1, Bo Zhang1, Ming Yi1, Chun‑Yue Weng1, Shan Lin1, Hui Wu2,3, Xiang‑Tian Qin2,3, Feng Xu2,3, Yi Teng2,3, Shui‑Jin Yuan2,3, Zhi‑Qiang Liu1* and Yu‑Guo Zheng1 Abstract Ophiocordyceps sinensis has been used as a traditional medicine or healthy food in China for thousands of years. Hirsutella sinensis was reported as the only correct anamorph of O. sinensis. It is reported that the laboratory‑grown H. sinensis mycelium has similar clinical efcacy and less associated toxicity compared to the wild O. sinensis. The research of the H. sinensis is becoming more and more important and urgent. To gain deeper insight into the biological and pharmacological mechanisms, we sequenced the genome of H. sinensis. The genome of H. sinensis (102.72 Mb) was obtained for the frst time, with > 99% coverage. 10,200 protein‑encoding genes were predicted based on the genome sequence. A detailed secondary metabolism analysis and structure verifcation of the main ingredients were performed, and the biosynthesis pathways of seven ingredients (mannitol, cordycepin, purine nucleotides, pyrimi‑ dine nucleotides, unsaturated fatty acid, cordyceps polysaccharide and sphingolipid) were predicted and drawn. Furthermore, infection process and mechanism of H. sinensis were studied and elaborated in this article. The enzymes involved in the infection mechanism were also predicted, cloned and expressed to verify the mechanism. The genes and proteins were predicted and annotated based on the genome sequence. The pathways of several active compo‑ nents in H. sinensis were predicted and key enzymes were confrmed. The work presented here would improve the understanding of the genetic basis of this organism, and contribute to further research, production and application of H. sinensis. Keywords: Hirsutella sinensis, Genome sequencing, Biosynthesis pathways of ingredients, Traditional Chinese medicine Introduction O. sinensis. Tere are high function similarities between Ophiocordyceps sinensis, a fungus that parasitizes Lepi- wild O. sinensis and cultured H. sinensis,both of them be doptera larvae, has been used as a traditional medicine used to treat weakness after sickness, lung and kidney- or healthy food in China for thousands of years (Zhang associated diseases and sexual dysfunction (Buenz et al. et al. 2012; Zhu et al. 1998). Te use of O. sinensis has a 2005; Chen et al. 2001; Li et al. 2006; Zhou et al. 2009). long history in traditional Chinese medicine and Tibetan Recently, it is found that H. sinensis has activities to mod- medicine (Sharma 2004; Winkler 2008). It has been ulate immune responses, inhibit tumor cell proliferation, reported that H. sinensis is the only correct anamorph of enhance hepatic function, regulate insulin sensitivity, decrease plasma cholesterol levels and modulate ster- oidogenesis (Cheung et al. 2009; Fu et al. 2018; He et al. *Correspondence: [email protected] 1 Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College 2013; Lu et al. 2019). of Biotechnology and Bioengineering, Zhejiang University of Technology, For thousands of years, O. sinensis can only be obtained Hangzhou 310014, China by feld collection, so wild O. sinensis was called as Full list of author information is available at the end of the article © The Author(s) 2020. 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AMB Expr (2020) 10:105 Page 2 of 19 ‘golden Chinese traditional medicine’ (Chen et al. 2017). pharmaceutical manufacturing or provide the theoretical In recent years, due to high demand and the excessive basis for the realization of sustainable resource use of H. excavation, the supply of O. sinensis is almost exhausted sinensis; improve the research of H. sinensis and obtain a in many areas (Huang et al. 2017). Because of its impor- more efcient processes of production. Te work present tant applications in the Chinese traditional medicine, it is here would aid in understanding and carrying out future increasingly urgent to carry out the genetic studies of O. research on the genetic basis of biology of this organism sinensis to discover valuable secondary metabolic func- and contribute to the further production and applica- tional genes, elucidate related metabolic pathways and to tion of H. sinensis. In this way, it would contribute to the pave foundation for development of new approaches for world-wide application of O. sinensis. pharmacological utilization. Investigation of O. sinensis in molecular and genetic levels also would provide new Materials and methods insight into this organism and aid in its development and Isolation and identifcation of H. sinensis retention (Zhang et al. 2018). Te O. sinensis samples were collected at the surface and Recently, H. sinensis mycelia have been reported to various depths at diferent locations in Yushu, Qinghai have similar clinical efcacy and less associated toxic- province. Te temperature on the sampling sites was ity compared with wild O. sinensis, and H. sinensis is 28–34 °C in dry seasons and 11–17 °C in wet seasons. expected to be an alternative of O. sinensis (Liu et al. Samples were cultured at least 16 h after collection and 2015). Te main biological and pharmacological active collected in sterile plastic containers. Subsequently, the ingredients in H. sinensis are mannitol, cordycepin, samples were cultured in saline, and then transported to purine nucleotides, pyrimidine nucleotides, unsaturated the laboratory by sterilized poly-ethylene bags. fatty acid, cordyceps polysaccharide and sphingolipid, In order to isolate H. sinensis, several isolation proce- which are consistent with the active ingredient in wild dures were carried out. Fresh and complete O. sinensis O. sinensis (Du et al. 2017; Olatunji et al. 2018). Tus, were frst selected out, and impurities on the surface of artifcially cultured mycelia of H. sinensis have become fruiting bodies were cleaned up by sterile water. Ten increasingly used in medicinal products (Li et al. 2019). fruiting bodies were washed several times with sterile In order to clarify the biological and pharmacological purifed water, and disinfection was carried out by con- mechanisms of O. sinensis, we carried out the genome ventional method by using 0.1% mercuric chloride. Sub- sequencing of H. sinensis, anamorph of O. sinensis, for sequently, worms and stromata were carefully separated the frst time, described the annotation and gene expres- by sterile scalpel in sterile conditions. Tree parts of the sion, and analyzed the complete sequencing data (Li et al. tissues were picked and cultured on the sterilized PDA 2016). Genome content, evolutionary analyses, and inves- slant medium at 16 °C. Growth condition was recorded tigation of some of the genetic features underlying the daily. In addition, worms and stromata were broken apart unique biology of H. sinensis have been defned according with sterile forceps, and center white mycelium tissues to the analysis from complete sequence data. Ten genes were seeded in PDA medium. and enzymes which control the biosynthesis of the active Te isolation media of potato dextrose agar (PDA; ingredients were obtained according to the sequenc- Difco, Detroit, MI, USA) was prepared and then auto- ing and annotation results. We further investigated claved at 115 °C for 30 min before use. Liquid PDA the interaction of diferent pathways and correspond- medium was composed 20% potatoes, 2.0 g/L glucose, ing enzymes, and tried to fnd out the real medicinal 0.46 g/L KH2PO4, 0.5 g/L MgSO4·H2O, 10.0 mg/L VB1, ingredients and how they work. Also, structure verif- 1.0 mg/L K2HPO4. Solid PDA medium needs another cation of the main ingredients was employed to clarify. 2% agar. Te fermentation medium: 1.0% glucose, 1.0% Te infection mechanism was investigated to reveal the molasses, 0.5% silkworm chrysalis powder, 1.0% soy- microbial properties of H. sinensis (Olatunji et al. 2018). bean meal, 0.5% yeast extract, 0.01% MgSO4·H2O, 0.02% Te genome sequence obtained in this study will facili- KH2PO4. tate understanding of the genetic basis of many traits at genome level and allow the undertaking of genome-wide Genome sequencing and analysis association studies of H. sinensis (Tang et al. 2015). Sev- High-quality genomic DNA was prepared from H. sinen- eral features of H. sinensis were investigated based on its sis using a standard phenol/chloroform method. To pre- genome. Te availability of genome will also facilitate the vent RNA and protein contamination, the prepared DNA identifcation and manipulation of candidate genes or was treated with RNase A and proteinase K. Te detailed genomic regions to generate the new ways to synthesize procedure of phenol/chloroform method was described new compounds with potentials in pharmaceuticals, and as follows: 0.5 g ground powder of H. sinensis was resus- pave foundation for development of new drugs for the pended in 1 mL TE bufer containing 110 μL 10% SDS, Jin et al.