Introduction

(—)-Huperzine A(Hup A，［(5R，9R，11E)-5-amino-11-ethylidene-5，6，9，10-tetrahydro-7-methyl-5，9-methano-cycloocteno［b］pyridine-2(1H)-one］，Figure 1)，is a competitive and reversible inhibitor of acetyl cholinesterase that is known to improve general cognitive function，global status，behavioral disturbance，and functional performance(Ha et al.，2011).It is found and extracted from a small group of the plant family Huperziaceae with a very low yield(0.0047 to 0.025%in Huperzia serrata)(Ma et al.，2005；Ha et al.，2011).These plants are not abundant and are only found in very specialized habitats(Ma et al.，2005).Tissue culture of the host plants is rarely successful，and the rigid molecular configuration of Hup A makes chemical synthesis difficult(Ha et al.，2011).A reliable and sustainable source is required due to the high demand for Hup A in Alzheimer's disease treatment.

After the discovery of the Taxol-producing endophytic fungus，endophytes have been the subject of increasing focus for many high-value bioactive secondary metabolites(SMs)(Venugopalan and Srivastava，2015).Their host plants have often become endangered or endemic due to excessive exploitation(Venugopalan and Srivastava，2015).Microbial fermentation has certain inherent advantages，such as simple and inexpensive culture，fast growth，ease of yield enhancement and scaling up，simple genetics，and ease of manipulation(Venugopalan and Srivastava，2015).Several Hup Aproducing endophytes，such as Shiraia sp.Slf14，Colletotrichum gloeosporioides ES026，Trichoderma sp.，and Paecilomycestenuis YS-13，have been isolated from H.serrata(Zhu et al.，2010；Wang et al.，2011；Zhao et al.，2013；Dong et al.，2014；Su and Yang，2015).However，few reports explore the mechanism of the production of host plant associated SMs in endophytes，instead screening the SM-producing strains(Venugopalan and Srivastava，2015).

The biosynthetic pathway of Hup A was proposed by Ma et al.in the Lycopodiaceae family，according to the chemical synthesis of lycopodium alkaloids(Figure 1)(Ma and Gang，2004).The proposed pathway starts with the decarboxylation of lysine(by lysine decarboxylase，LDC)，which is then transformed toΔ1-piperideine via 5-amiopentanal(by copper amine oxidase，CAO).At the same time，two malonyl-CoA are condensed to form acetonedicarboxylic acid(perhaps by polyketide synthase，PKS).Δ1-piperideine is then coupled to acetonedicarboxylic acid(or its bisCo A ester)to form 4-(2-piperidyl)acetoacetate(4PAA)(or 4-(2piperidyl)acetoacetyl-CoA，4PAACo A).4PAA/4PAACoA is then decarboxylated(4PAACo A is perhaps hydrolyzed first)to form pelletierine.Pelletierine and 4PAA/4PAACo A，or some derivatives，are coupled，accompanied by requisite decarboxylation，to form phlegnarine.After oxidative ring closure of phlegnarine to form lycodane(likely by berberine bridge enzyme，BBE)，other oxidative modifications lead a series of precursors to Hup A，presumably catalyzed by cytochrome P450s or 2-oxoglutarate-dependent dioxygenases(2OGD)(Ma and Gang，2004).However，among these putative Hup A biosynthetic genes，only LDC，CAO，and PKS genes were cloned，characterized and expressed in vitro(Sun et al.，2012；Yang et al.，2016；Xu et al.，2017).None of these putative biosynthetic genes was confirmed by in vivo experiments to establish a direct relationship with Hup A.It has long been existing of the conflicts about the source of the endophytic biosynthetic genes(Venugopalan and Srivastava，2015).Fungal genes showed homology to the plant counterparts，supporting the horizontal gene transfer(HGT)theory(Staniek et al.，2009；Zhang et al.，2009)，while low similarity was found in fungi with the corresponding plant genes，supporting co-evolution rather than HGT(Hedden et al.，2001；Yang et al.，2014).Up to now，there has been no systematic analysis of the Hup A biosynthesis mechanism in endophytic fungi，nor of the origin.

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Figure 1　The proposed biosynthetic pathway of Hup A in H.serrata

LDC：lysine decarboxylase；CAO：copper amine oxidase；PKS：polyketides synthases；BBE：berberine bridge enzyme；2OGD：2-oxoglutarate-dependent dioxygenases.

In most cases，the genes coding for SM-biosynthesis enzymes are clustered in fungal genomes and silenced by heterochromatic histone marks(Keller and Hohn，1997).Typical heterochromatic marks are characterized by both hypoacetylation of the histone tails and methylation of histone H3 on lysine 9 or lysine 27(Bannister et al.，2001；Nakayama et al.，2001；Shahbazian and Grunstein，2007；Pettit，2011).The“closed”heterochromatic structures could be reversed during SM activation.In filamentous fungi，chromatin structure and function has only been studied in a few model systems，such as Neurospora crassa，Aspergillus nidulans，and Penicillium chrysogenum(Strauss and Reyes-Dominguez，2011).The first evidence of chromatin involvement in SM regulation was that HDAC deletion leads to transcriptional activation of two telomere-proximal SM gene clusters and to an elevated level of sterigmatocystin and penicillin in A.nidulans(Shwab et al.，2007).Subsequently，it was found that during the active growth phase，the silent sterigmatocystin gene cluster is marked by H3K9me3 and contains high levels of the heterochromatin protein-1(Hep A)(Reyes-Dominguez et al.，2010).Upon growth arrest and activation of SM，Hep A and trimethylated H3K9 levels decrease concomitantly with increasing levels of acetylated histone H3(Reyes-Dominguez et al.，2010).However，there has yet been no report on the epigenetic regulation of SM production in endophytes.

In this study，we sequenced the whole genomes of two Hup A-producing endophytes(Penicillium polonicum hy4 and Colletotrichum gloeosporioides Cg01)from H.serrata and compared the differentially expressed genes(DEGs)between the induced group(with added extracts of H.serrata)and a control group.We characterized the whole genomes and obtained the KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment pathways and GO(Gene Ontology)enrichment function clusters of these DEGs.The focus of this study is the genes proposed in plants to biosynthesize Hup A，which include LDC，CAO，PKS，BBE，P450，and 2OGD genes，as well as the epigenetic modification genes.It is the first report on epigenetic modification analysis in high value SM-producing endophytic fungi.This study sheds new light on Hup A biosynthesis and regulation in Hup A-producing endophytic fungi and is the basis of further synthetic biology study for industrial production of Hup A from fungi.