Lycopodium Alkaloids from Huperzia goebelii.

One new fawcettimine-type Lycopodium alkaloid, hupertimine F (1), together with five known (2-6) Lycopodium alkaloids were isolated from Huperzia goebelii. The structure of 1 was elucidated by 1D and 2D NMR spectra, HRESIMS, and X-ray diffraction. Structurally, 1 represents the fourth example of Lycopodium alkaloids characterized by a 5/5/5/5/6 pentacyclic ring system with a 1-aza-7-oxabicyclo[2.2.1]heptane moiety. These known compounds 2, 3, 5, and 6 were isolated from H. goebelii for the first time. Compounds 1-6 were evaluated for acetylcholinesterase, butyrylcholinesterase and monoamine oxidase B inhibitory activities in vitro.

[Mining and identification of a biosynthetic gene cluster producing xanthocillin analogues from Penicillium chrysogenum MT-40, an endophytic fungus of Huperzia serrata].

Xanthocillin is a unique natural product with an isonitrile group and shows remarkable antibacterial activity. In this study, the genome of an endophytic fungus Penicillium chrysogenum MT-40 isolated from Huperzia serrata was sequenced, and the gene clusters with the potential to synthesize xanthocillin analogues were mined by local BLAST and various bioinformatics analysis tools. As a result, a biosynthetic gene cluster (named for) responsible for the biosynthesis of xanthocillin analogues was identified by further heterologous expression of the key genes in Aspergillus oryzae NSAR1. Specifically, the ForB catalyzes the synthesis of 2-formamido-3-(4-hydroxyphenyl) acrylic acid, and the ForG catalyzes the dimerization of 2-formamido-3-(4-hydroxyphenyl) acrylic acid to produce the xanthocillin analogue N, N'-(1, 4-bis (4-hydroxyphenyl) buta-1, 3-diene-2, 3-diyl) diformamide. The results reported here provide a reference for further discovery of xanthocillin analogues from fungi.

Assembly and phylogenetic analysis of the mitochondrial genome of endangered medicinal plant Huperzia crispata.

Huperzia crispata is a traditional Chinese herb plant and has attracted special attention in recent years for its products Hup A can serve as an acetylcholinesterase inhibitor (AChEI). Although the chloroplast (cp) genome of H. crispata has been studied, there are no reports regarding the Huperzia mitochondrial (mt) genome since the previously reported H. squarrosa has been revised as Phlegmariurus squarrosus. The mt genome of H. crispata was sequenced using a combination of long-read nanopore and Illumina sequencing platforms. The entire H. crispata mt genome was assembled in a circular with a length of 412,594 bp and a total of 91 genes, including 45 tRNAs, 6 rRNAs, 37 protein-coding genes (PCGs), and 3 pseudogenes. Notably, the rps8 gene was present in P. squarrosus and a pseudogene rps8 was presented in H. crispata, which was lacking in most of Pteridophyta and Gymnospermae. Intron-encoded maturase (mat-atp9i85 and mat-cobi787) genes were present in H. crispata and P. squarrosus, but lost in other examined lycophytes, ferns, and Gymnospermae plants. Collinearity analysis showed that the mt genome of H. crispata and P. squarrossus is highly conservative compared to other ferns. Relative synonymous codon usage (RSCU) analysis showed that the amino acids most frequently found were phenylalanine (Phe) (4.77%), isoleucine (Ile) (4.71%), lysine (Lys) (4.26%), while arginine (Arg) (0.32%), and histidine (His) (0.42%) were rarely found. Simple sequence repeats (SSR) analysis revealed that a total of 114 SSRs were identified in the mt genome of H. crispata and account for 0.35% of the whole mt genome. Monomer repeats were the majority types of SSRs and represent 91.89% of the total SSRs. In addition, a total of 1948 interspersed repeats (158 forward, 147 palindromic, and 5 reverse repeats) with a length ranging from 30 bp to 14,945 bp were identified in the H. crispata mt genome and the 30-39-bp repeats were the most abundant type. Gene transfer analysis indicated that a total of 12 homologous fragments were discovered between the cp and mt genomes of H. crispata, accounting for 0.93% and 2.48% of the total cp and mt genomes, respectively. The phylogenetic trees revealed that H. crispata was the sister of P. squarrosus. The Ka/Ks analysis results suggested that most PCGs, except atp6 gene, were subject to purification selection during evolution. Our study provides extensive information on the features of the H. crispata mt genome and will help unravel evolutionary relationships, and molecular identification within lycophytes.

Lycopodium Alkaloids from Huperzia serrata and Their Anti-acetylcholinesterase Activities.

One new fawcettimine-type alkaloid (1), one new miscellaneous-type alkaloid (2), four new lycodine-type alkaloids (3-6), and eight known ones (7-14) were isolated from the whole plants of Huperzia serrata. Their structures and absolute configurations were elucidated based on spectroscopic data, X-ray diffraction, ECD calculation and Mosher's method. Compound 1 was a rare C18 N2 -type Lycopodium alkaloid, possessing serratinine skeleton with an amide side chain in C-5. The absolute configuration of the 18-OH of compounds 4-6 were first determined by Mosher's method. Moreover, compounds 1-14 were assayed anti-acetylcholinesterase effect in vitro, and compound 7 showed significant anti-acetylcholinesterase activity with an IC50 value of 16.18±1.64 µM.

New insights into the composition and diversity of endophytic bacteria in cultivated Huperzia serrata.

Endophytic bacteria play crucial roles in the growth and bioactive compound synthesis of host plants. In this study, the composition and diversity of endophytic bacteria in the roots, stems, and leaves from 3-year-old artificially cultivated Huperzia serrata were investigated using Illumina HiSeq sequencing technology. Total effective reads were assigned to 936 operational taxonomic units (OTUs), belonging to 12 phyla and 289 genera. A total of 28, 3, and 2 OTUs were exclusive to the roots, stems, and leaves, respectively. The bacterial richness and diversity in the roots were significantly lower than those in the leaves and stems. The dominant genera with significant distribution differences among these plant tissue samples were Burkholderia-Caballeronia-Paraburkholderia, Sphingomonas, Acidibacter, Bradyrhizobium, Bryobacter, Methylocella, Nocardioides, Acidothermus, and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium. Furthermore, the differences in the bacterial communities associated with these plant tissue samples were visualized using principal coordinate analysis and cluster pedigree diagrams. Linear discriminant analysis effect size explained statistically significant differences among the endophytic bacterial microbiota in these plant tissue samples. Overall, this study provides new insights into the diversity and distribution patterns of endophytic bacteria in the different tissues of H. serrata.

Anti-neuroinflammatory effects of alkaloid-enriched extract from Huperzia serrata on lipopolysaccharide-stimulated BV-2 microglial cells.

CONTEXT: Alkaloid-enriched extract of Huperzia serrata (Thunb.) Trevis (Lycopodiaceae) (HsAE) can potentially be used to manage neuronal disorders. OBJECTIVE: This study determines the anti-neuroinflammatory effects of HsAE on lipopolysaccharide (LPS)-stimulated BV-2 microglial cells and the underlying mechanisms. MATERIALS AND METHODS: BV-2 cells were pre- or post-treated with different concentrations of HsAE (25-150 µg/mL) for 30 min before or after LPS induction. Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and no cytotoxicity was found. Nitric oxide (NO) concentration was determined using Griess reagent. The levels of prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 were determined using enzyme-linked immunosorbent assay. The levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 and the phosphorylation of mitogen-activated protein kinase (MAPK) were analyzed using western blotting. RESULTS: HsAE reduced LPS-induced NO production with half-maximal inhibitory concentration values of 99.79 and 92.40 µg/mL at pre- and post-treatment, respectively. Pre-treatment with HsAE at concentrations of 50, 100, and 150 µg/mL completely inhibited the secretion of PGE2, TNF-α, IL-6, and IL-1ß compared to post-treatment with HsAE. This suggests that prophylactic treatment is better than post-inflammation treatment. HsAE decreased the expression levels of iNOS and COX-2 and attenuated the secretion of pro-inflammatory factors by downregulating the phosphorylation of p38 and extracellular signal-regulated protein kinase in the MAPK signaling pathway. DISCUSSION AND CONCLUSIONS: HsAE exerts anti-neuroinflammatory effects on LPS-stimulated BV-2 cells, suggesting that it may be a potential candidate for the treatment of neuroinflammation in neurodegenerative diseases.

Neuroprotective effects of total alkaloids fraction of Huperzia serrata on scopolamine-induced neurodegenerative animals.

Huperzia serrata contains Huperzine A (HupA)-an alkaloid used to treat cognitive dysfunction. In this study, we used the total alkaloids (HsAE) to investigate their potential in managing cognitive impairment in comparison with HupA. The antioxidant activity was measured by DPPH assay. In the cellular study, the cell viability and level of ACh of SH-SY5Y cells were evaluated after pretreated with HsAE and scopolamine. For in vivo assay, mice were pre-treated with HsAE, and HupA and undergone scopolamine injection for cognitive impairment. The behavioral tests including the Y-maze and Morris water maze test and the AChE activity, the SOD, CAT, MDA level in the hippocampus and cortex were evaluated. HsAE showed significant scavenging properties on DPPH radicals. HsAE was not toxic to SH-SY5Y cells, and can rescue these cells upon scopolamine treatment. Intriguingly, HsAE showed the neuroprotection against scopolamine-induced amnesia in mice. Moreover, HsAE decreased AChE activity, MDA level, increased antioxidative enzyme activity in the hippocampus as well as cortex of mice, which was relatively better than that of HupA. These findings suggested that HsAE may significantly protect the neurons of mice with scopolamine-induced memory impairment connected to AChE depletion and oxidative stress.

The temporal and spatial endophytic fungal community of Huperzia serrata: diversity and relevance to huperzine A production by the host.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

The complete chloroplast genome of the medical plant Huperzia crispata from the Huperziaceae family: structure, comparative analysis, and phylogenetic relationships.

BACKGROUND: Huperzia crispata, belonging to the Huperziaceae family, is one of the most essential resources of huperzine A for candidate drugs to treat Alzheimer's diseases. However, there is very limited information about H. crispat, and its taxonomic status and interspecific relationships between Huperzia species are still unclear. To investigate the taxonomic classification of Huperzia species and identify species discrimination markers, the complete chloroplast (cp) genome of H. crispata was sequenced and characterized for the first time. METHODS AND RESULTS: Total genomic DNA was isolated and sequenced using the next-generation Illumina NovaSeq 6000 platform. The data were filtered, assembled and annotated by a series software and web service. The results were as follows: the cp genome of H. crispata was 154,320 bp long with a large single-copy (LSC) region of 104,023 bp, a small single-copy (SSC) region of 19,671 bp, and a pair of inverted repeat (IRa and IRb) regions of 15,313 bp. A total of 131 genes, including 87 protein-coding genes, 36 transfer RNA genes (tRNAs), and eight ribosome RNA genes (rRNAs), were annotated in the cp genome. The contraction and expansion of the inverted repeat (IR) regions were relatively conserved in the Huperzia genus. Codon usage bias analysis showed that the encoding rate at the 3-end of codon A/T (74.34%) was significantly higher than that of C/G (25.66%). A total of 8 hotspot loci with high Pi values (> 0.06) were identified in the four Huperzia species based on nucleic acid diversity analysis. Ka/Ks selective pressure analysis demonstrated that the cemA gene is the most common gene undergoing positive selection among Huperzia. In addition, a total of 261 simple sequence repeats and 179 interspersed repeats were identified in the cp genome. Phylogenetic tree analysis based on the complete protein sequences of 23 related species of H. crispata indicated that H. serrata f. longipetiolata is a sister of H. crispata, suggesting that H. serrata f. longipetiolata and H. crispata are more closely related than H. serrata and H. lucidula. CONCLUSIONS: The results strongly supported that H. crispata was more closely related to H. serrata f. longipetiolata than to H. serrata and H. lucidula within the Huperzia genus. The outcome provided important information for the phylogenetic analysis of the subsequent specific molecular species identification in Huperzia. The present results will provide valuable information for further research into the classification, phylogeny and species identification of Huperzia plants.

Diversity of endophytic fungal community in Huperzia serrata from different ecological areas and their correlation with Hup A content.

BACKGROUND: Huperzine A (Hup A) has attracted considerable attention as an effective therapeutic candidate drug used to treat Alzheimer's disease. Whereas, the production of Hup A from wild plants faced a major challenge, which is the wild Huperzia Serrata harbor a low Hup A content, has a long-life cycle, and has a small yield. At present, several reports showed that Hup A is produced by various endophytic fungal strains isolated from H. serrata, thereby providing an alternative method to produce the compound and reduce the consumption of this rare and endangered plant. However, till now, very few comprehensive studies are available on the biological diversity and structural composition of endophytic fungi and the effects of endophytic fungi on the Hup A accumulation in H. serrata. RESULTS: In this research, the composition and diversity of fungal communities in H. serrata were deciphered based on high-throughput sequencing technology of fungal internal transcribed spacer regions2 (ITS2). The correlation between endophytic fungal community and Hup A content was also investigated. Results revealed that the richness and the diversity of endophytic fungi in H. serrata was various according to different tissues and different ecological areas. The endophytic fungal communities of H. serrata exhibit species-specific, ecological-specific, and tissue-specific characteristics. There are 6 genera (Ascomycota_unclassified, Cyphellophora, Fungi_unclassified, Sporobolomyces, and Trichomeriaceae_unclassified) were significantly positively correlated with Hup A content in all two areas, whereas, there are 6 genera (Auricularia, Cladophialophora, Cryptococcus, Mortierella, and Mycena) were significantly negatively correlated with Hup A content of in all two areas. CONCLUSIONS: This study indicated a different composition and diverse endophytic fungal communities in H. serrata from different organs and ecological areas. The current study will provide the realistic basis and theoretical significance for understanding the biological diversity and structural composition of endophytic fungal communities in H. serrata, as well as providing novel insights into the interaction between endophytic fungi and Hup A content.

Delineating biosynthesis of Huperzine A, A plant-derived medicine for the treatment of Alzheimer's disease.

Huperzine A (HupA) is a plant-derived lycopodium alkaloid used for the treatment of Alzheimer's disease due to its inhibition against acetylcholinesterase. Currently, industrial production of HupA relies primarily on direct extraction from Huperzia serrate, a perennial herbaceous plant. However, this strategy cannot satisfy the increasing demand for HupA due to scarcity of H. serrate whose growth is quite slow. Pathway engineering has emerged as a novel strategy for the production of HupA. Unfortunately, the biosynthesis mechanism of HupA has not been well documented. In this review, we summarize not only the methods for plant extraction and chemical synthesis but also state-of-the-art advances in biosynthesis of HupA, including synthetic pathways, key enzymes, and especially catalytic mechanisms. Overall, this review aims to provide valuable insights for complete biosynthesis of Hup A.

Hupertimines A-E, Fawcettimine-Type Lycopodium Alkaloids from Huperzia serrata.

Five new fawcettimine-type Lycopodium alkaloids, hupertimines A-E (1-5), were discovered from the whole plant of Huperzia serrata, along with two known alkaloids, 8α-hydroxyphlegmariurine B (6) and 8ß-hydroxyphlegmariurine B (7). The structures of 1-7 were identified through HR-MS, IR, 1 H, 13 C, and 2D NMR, and single-crystal X-ray diffraction analysis. Structurally, compound 1 was the fourth example of Lycopodium alkaloid with an ether linkage between C-5 and C-13 and 2 was the third example of Lycopodium alkaloid with a 5/5/5/5/6 pentacyclic ring system and featuring a 1-aza-7-oxabicyclo[2.2.1]heptane unit. Compounds 1-7 were tested for their BACE1 inhibitory activity. In addition, the correct 1 H- and 13 C-NMR data for 7 were reported in current study.

Two new diterpenoids from Penicillium chrysogenum MT-12, an endophytic fungus isolated from Huperzia serrata.

Two new diterpenoids, penicichrysogene A (1) and penicichrysogene B (2), were isolated from the solid substrate fermentation cultures of Penicillium chrysogenum MT-12, an endophytic fungus isolated from the medicinal plant of Huperzia serrata. Their structures were elucidated on the basis of extensive spectroscopic and spectrometric data (1D and 2D NMR, UV, IR, and HRESIMS). The absolute configurations of 1 and 2 were assigned on the basis of experimental and calculated electronic circular dichroism spectra. Compound 1 exhibited inhibitory activity on ATP release of thrombin-activated platelets with IC50 = 42.7 ± 3.5 µM.

Huperzine A and Its Neuroprotective Molecular Signaling in Alzheimer's Disease.

Huperzine A (HupA), an alkaloid found in the club moss Huperzia serrata, has been used for centuries in Chinese folk medicine to treat dementia. The effects of this alkaloid have been attributed to its ability to inhibit the cholinergic enzyme acetylcholinesterase (AChE), acting as an acetylcholinesterase inhibitor (AChEI). The biological functions of HupA have been studied both in vitro and in vivo, and its role in neuroprotection appears to be a good therapeutic candidate for Alzheimer´s disease (AD). Here, we summarize the neuroprotective effects of HupA on AD, with an emphasis on its interactions with different molecular signaling avenues, such as the Wnt signaling, the pre- and post-synaptic region mechanisms (synaptotagmin, neuroligins), the amyloid precursor protein (APP) processing, the amyloid-ß peptide (Aß) accumulation, and mitochondrial protection. Our goal is to provide an integrated overview of the molecular mechanisms through which HupA affects AD.

Secondary metabolites of endophytic fungi isolated from Huperzia serrata.

The natural product Huperzine A isolated from Huperzia serrata is a targeted inhibitor of acetylcholinesterase that has been approved for clinical use in the treatment of Alzheimer's disease. Given the large demand for natural sources of Huperzine A  (Hup. A), efforts have been made to explore whether it is also produced by endophytic fungi from H. serrata and, if so, identify its biosynthetic pathway. These studies have indicated that endophytic fungi from H. serrata represent a huge and largely untapped resource for natural products (including Hup. A) with chemical structures that have been optimized by evolution for biological and ecological relevance. To date, more than three hundred endophytic fungi have been isolated from H. serrata, of which 9 strains can produce Hup. A, whilst more than 20 strains produce other important metabolites, such as polyketones, xanthones, alkaloids, steroids, triterpenoids, furanone derivatives, tremulane sesquitepenes and diterpenoids. In total, 200 secondary metabolites have been characterized in endophytic fungi from H. serrata to date. Functionally, some have cholinesterase-inhibitory or antibacterial activity. This review also considers the different classes of secondary metabolites produced by endophytic fungi, along with their possible applications. We systematically describe the taxonomy, biology, and chemistry of these secondary metabolites. It also summarizes the biosynthetic synthesis of metabolites, including that of Hup. A. The review will aid researchers in obtaining a clearer understanding of this plant-endophyte relationship to better exploit the excellent resources it offers that may be utilized by pharmaceutical industries.

Simultaneous separation and determination of three huperzine alkaloids in Huperzia serrata and its preparations by cyclodextrin-modified mixed micellar electrokinetic capillary chromatography.

In this study, a simple and sensitive cyclodextrin-modified mixed micellar electrokinetic capillary chromatography (CD-MEKC) method has been developed for the simultaneous separation and determination of Huperzine A (HupA), Huperzine B (HupB) and Huperzine C (HupC) in Huperzia serrata (H. serrata). The optimal conditions (pH 9.3) were composed of 10 mM sodium tetraborate solution, 40 mM sodium dodecyl sulfate (SDS), 50 mM sodium cholate (SC) and 3.0 mM mono-(6-ethylenediamine-6-deoxy)-ß-cyclodextrin (ED-ß-CD). The separation and determination process were performed on a P/ACE MDQ capillary electrophoresis system, the separation voltage was 15 kV, the temperature was 25 °C and the detection wavelength was 308 nm. Under the optimum conditions, the migration time was less than 9 min. The LOD and LOQ were between 0.38 and 0.80 µg/mL and 1.2-2.3 µg/mL, respectively. The developed method, with excellent precision and accuracy, was applied for the determination of three alkaloids in H. serrata and its formulations.

Endophytic Fungal Community of Huperzia serrata: Diversity and Relevance to the Production of Huperzine A by the Plant Host.

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.

Metabolites from the endophytic fungus colletotrichum sp. F168.

An endophytic fungus, Colletotrichum sp. F168, was isolated from plant Huperzia serrata (Thunb. ex Murray) Trev. and was subjected to phytochemical investigation. Nine compounds including two new structures were obtained from SDA solid fermentation products of strain F168, which were elucidated by extensive spectroscopic analyses, including 1 D- and 2 D-NMR, and HR-MS experiments. The acetylcholinesterase inhibitory activity of two new compounds 1-2 was tested in vitro. Two new compounds didn't show evident acetylcholinesterase inhibitory activity.

Response surface methodology-mediated improvement of the irradiated endophytic fungal strain, Alternaria brassicae AGF041 for Huperzine A-hyperproduction.

Huperzine A (HupA) is an anti-Alzheimer's therapeutic and a dietary supplement for memory boosting that is extracted mainly from Huperziacae plants. Endophytes represent the upcoming refuge to protect the plant resource from distinction but their HupA yield is still far from commercialization. In this context, UV and gamma radiation mutagenesis of the newly isolated HupA-producing Alternaria brassicae AGF041 would be applied in this study for improving the endophytic HupA yield. Compared to non-irradiated cultures, UV (30-40 min, exposure) and γ (0·5 KGy, dose) irradiated cultures, each separately, showed a significant higher HupA yield (17·2 and 30·3%, respectively). While, application of a statistically optimized compound irradiation (0·70 KGy of γ treatment and 42·49 min of UV exposure, sequentially) via Response Surface Methodology (RSM) resulted in 53·1% production increase. Moreover, a stable selected mutant strain CM003 underwent batch cultivation using a 6·6 l bioreactor for the first time and was successful for scaling up the HupA production to 261·6 µg l-1 . Findings of this research are demonstrated to be valuable as the employed batch fermentation represents a successful starting step towards the promising endophytic HupA production at an industrial scale.

Illumina-based analysis yields new insights into the diversity and composition of endophytic fungi in cultivated Huperzia serrata.

Endophytic fungi play an important role in plant growth. The composition and structure of endophytes vary in different plant tissues, which are specific habitats for endophyte colonization. To analyze the diversity and structural composition of endophytic fungi from toothed clubmoss (Huperzia serrata) that was artificially cultivated for 3 years, we investigated endophytic fungi from the roots, stems and leaves using comparative sequence analysis of the ITS2 region of the fungal rRNA genes sequenced with high-throughput sequencing technology. Seven fungal phyla were identified, and fungal diversity and structure varied across different tissues, with the most distinctive community features found in the roots. A total of 555 operational taxonomic units (OTUs) were detected, and 198 were common to all samples, and 43, 16, 16 OTUs were unique to the root, stem, leaf samples, respectively. Taxonomic classification showed that Ascomycota and Basidiomycota were dominant phyla, and Cladosporium, Oidiodendron, Phyllosticta, Sebacina and Ilyonectria were dominant genera. The relative abundance heat map at the genus level suggested that H. serrata had characteristic endophytic fungal microbiomes. Line discriminant analysis effect size analysis and principal coordinate analysis demonstrated that fungal communities were tissue-type and tissue-site specific. Overall, our study provides new insights into the complex composition of endophytic fungi in H. serrata.