HupA Production Research Articles

New and potent production platform of the acetylcholinesterase inhibitor huperzine A by gamma-irradiated Alternaria brassicae under solid-state fermentation.

Huperzine-A (HupA) is an emerging, powerful, and promising natural acetylcholinesterase inhibitor. Despite that, the achieved yields of HupA from microbial sources are still far from the industrial applications. Accordingly, this paper was conducted to valorize solid-state fermentation (SSF) as an efficient production platform of HupA. Four agro-industrial wastes, namely rice bran, potato peel, sugarcane bagasse, and wheat bran, were tested and screened as cultural substrates for the production of HupA by the endophytic Alternaria brassica under SSF. Maximum HupA production was attained on using rice bran moistened by Czapex's dox mineral broth. In the effort to increase the HupA titer, supplementation of the best moistening agent by different carbon and nitrogen sources was successfully investigated. Additionally, factors affecting HupA production under SSF including substrate concentration, moistening level, and inoculum concentration were optimized using response surface methodology. A Box-Behnken design was applied for generating a predictive model of the interactions between these factors. Under the optimum conditions of 15g rice bran, inoculum concentration of 5 × 106 spores mL-1, and 60% moisture level, HupA concentration was intensified to 518.93μgg-1. Besides, HupA production by the fungal strain was further enhanced using gamma-irradiation mutagenesis. The final HupA production was significantly intensified following exposure to 0.5 KGy gamma radiation to 1327μgg-1, which represents a 12.85-fold increase. This is the first report on the successful production of the natural fungal metabolite HupA under SSF. Moreover, the achieved yield in this study using agro-industrial wastes may contribute to reducing the cost of HupA manufacture.Key points• Different agro-industrial by-products were tried as cultural substrates for the production of the acetylcholinesterase inhibitor HupA under SSF for the first time.• Factors affecting HupA production under SSF were optimized using response surface methodology.• The final HupA production was intensified following exposure to gamma radiation recording 1327μgg-1, which represents a 12.85-fold increase.

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.

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 (30-40min, exposure) and γ (0·5KGy, 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 42·49min 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 6·6l bioreactor for the first time and was successful for scaling up the HupA production to 261·6µgl-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.

Isolation of endophytic fungi and screening of Huperzine A\u2013producing fungus from Huperzia serrata in Vietnam

Huperzine A (HupA), a natural Lycopodium alkaloid derived from Huperzia serrata (Thunb. ex Murray) Trev. plants, is a highly active acetylcholinesterase inhibitor and a key compound used for treating Alzheimer’s disease (AD). Recently, HupA has been reported in various endophytic fungi isolated from H. serrata. In the present study, 153 endophytic fungi were isolated from healthy tissues of H. serrata collected from natural populations in Lam Dong province of Central Vietnam. The endophytic fungi were identified based on morphological characteristics and Internal Transcribed Spacer sequences. Among them, 34 strains were classified into seven genera belonging to Ascomycota, including Alternaria, Fusarium, Trichoderma, Penicillium, Paecilomyces, and Phoma, and eight strains belonging to the genus Mucor (Zygomycota). The other strains remained unidentified. According to the results of thin-layer chromatography and high-performance liquid chromatography, only one of the 153 strains, Penicillium sp. LDL4.4, could produce HupA, with a yield 1.38 mg l−1 (168.9 µg g−1 dried mycelium) when cultured in potato dextrose broth, which was considerably higher than that of other reported endophytic fungi. Such a fungus is a promising candidate and alternative to presently available HupA production techniques for treating AD and preventing further memory decline.

Production and enhancement of the acetylcholinesterase inhibitor, huperzine A, from an endophytic Alternaria brassicae AGF041.

Huperzine A (HupA) is a potent acetylcholinesterase (AChE) inhibitor of a great consideration as a prospective drug candidate for Alzheimer's disease treatment. Production of HupA by endophytes offers an alternative challenge to reduce the massive plant harvest needed to meet the increasing demand of HupA. In the current study, some endophytic fungal and actinobacterial isolates from the Chinese herb, Huperzia serrata, underwent liquid fermentation, alkaloid extraction, and screening for AChE inhibition and HupA production. Among these isolates, Alternaria brassicae AGF041 strain was the only positive strain for HupA production with the maximum AChE inhibition of 75.5%. Chromatographic analyses verified the identity of the produced HupA. The HupA production was efficiently maximized up to 42.89μg/g of dry mycelia, after optimization of thirteen process parameters using multifactorial statistical approaches, Plackett-Burman and central composite designs. The statistical optimization resulted in a 40.8% increase in HupA production. This is the first report to isolate endophytic actinobacteria with anti-AChE activity from H. serrata, and to identify an endophytic fungus A. brassicae as a new promising start strain for a higher HupA yield.

Genomic Characterization Provides New Insights Into the Biosynthesis of the Secondary Metabolite Huperzine a in the Endophyte Colletotrichum gloeosporioides Cg01.

A reliable source of Huperzine A (HupA) meets an urgent need due to its wide use in Alzheimer's disease treatment. In this study, we sequenced and characterized the whole genomes of two HupA-producing endophytes, Penicillium polonicum hy4 and Colletotrichum gloeosporioides Cg01, to clarify the mechanism of HupA biosynthesis. The whole genomes of hy4 and Cg01 were 33.92 and 55.77 Mb, respectively. We compared the differentially expressed genes (DEGs) between the induced group (with added extracts of Huperzia serrata) and a control group. We focused on DEGs with similar expression patterns in hy4 and Cg01. The DEGs identified in GO (Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were primarily located in carbon and nitrogen metabolism and nucleolus, ribosome, and rRNA processing. Furthermore, we analyzed the gene expression for HupA biosynthesis genes proposed in plants, which include lysine decarboxylase (LDC), copper amine oxidase (CAO), polyketides synthases (PKS), etc. Two LDCs, one CAO, and three PKSs in Cg01 were selected as prime candidates for further validation. We found that single candidate biosynthesis-gene knock-out did not influence the HupA production, while both LDC gene knock-out led to increased HupA production. These results reveal that HupA biosynthesis in endophytes might differ from that proposed in plants, and imply that the HupA-biosynthesis genes in endophytic fungi might co-evolve with the plant machinery rather than being acquired through horizontal gene transfer (HGT). Moreover, we analyzed the function of the differentially expressed epigenetic modification genes. HupA production of the histone acetyltransferase (HAT) deletion mutant ΔCgSAS-2 was not changed, while that of the histone methyltransferase (HMT) and histone deacetylase (HDAC) deletion mutants ΔCgClr4, ΔCgClr3, and ΔCgSir2-6 was reduced. Recovery of HupA-biosynthetic ability can be achieved by retro-complementation, demonstrating that HMT and HDACs associated with histone modification are involved in the regulation of HupA biosynthesis in endophytic fungi. This is the first report on epigenetic modification in high value secondary metabolite- producing endophytes. These findings shed new light on HupA biosynthesis and regulation in HupA-producing endophytes and are crucial for industrial production of HupA from fungi.

Identification and characterization of L-lysine decarboxylase from Huperzia serrata and its role in the metabolic pathway of lycopodium alkaloid

Lysine decarboxylation is the first biosynthetic step of Huperzine A (HupA). Six cDNAs encoding lysine decarboxylases (LDCs) were cloned from Huperzia serrata by degenerate PCR and rapid amplification of cDNA ends (RACE). One HsLDC isoform was functionally characterized as lysine decarboxylase. The HsLDC exhibited greatest catalytic efficiency (kcat/Km, 2.11 s−1 mM−1) toward L-lysine in vitro among all reported plant-LDCs. Moreover, transient expression of the HsLDC in tobacco leaves specifically increased cadaverine content from zero to 0.75 mg per gram of dry mass. Additionally, a convenient and reliable method used to detect the two catalytic products was developed. With the novel method, the enzymatic products of HsLDC and HsCAO, namely cadaverine and 5-aminopentanal, respectively, were detected simultaneously both in assay with purified enzymes and in transgenic tobacco leaves. This work not only provides direct evidence of the first two-step in biosynthetic pathway of HupA in Huperzia serrata and paves the way for further elucidation of the pathway, but also enables engineering heterologous production of HupA.

Isolation and characterzation of endophytic Huperzine A-producing fungi from Phlegmariurus phlegmaria

Based on special colony morphology, 340 endophytic fungi were isolated from the stems, leaves, and roots of Phlegmariurus phlegmaria. Among the 340 strains, eight, representative species (MY298, MJ216, MY237, MJ422, MY311, MY183, MJ484, and MY252) could produce HupA analyzed by UPLC-MS/MS (Ultra performance liquid chromatography), which compared with standard HupA under the same chromatographic conditions. And through the standard curve generated, among these HupA-producing fungi, the MY311 had highest content which was 40.53 µg/L. The extract of eight strains was examined by AChE inhibition activity assay in vitro and ELISA (enzyme-linked immunosorbent assay). These eight strains were found to have positive results for HupA production and MY311 extract obviously exhibited the highest inhibition activity. Furthermore, the genera of the eight fungi were identified based on the internal transcribed spacer sequence analysis. All eight distinctive genotypes were grouped into four fungal genera and six fungal species, however, MY311 and MY183 were Ceriporia lacerata and Hypoxylon investiens, which had never been reported as HupA-producing endophytic fungi.

Efficient strategy for maintaining and enhancing the huperzine A production of Shiraia sp. Slf14 through inducer elicitation.

Huperzine A (HupA), a naturally occurring lycopodium alkaloid, is a potent, highly specific and reversible inhibitor of acetylcholinesterase and is a potential treatment for Alzheimer's disease. However, isolating HupA from Huperziaceae plants is inefficient; thus, extracting this compound from endophytic fungi may be more controllable and sustainable. However, the large-scale production of this chemical from endophytes is limited by the innate instability of endophytic fungi. In this study, we maintained the stability and viability of the HupA-producing endophytic fungus Shiraia sp. Slf14 and enhanced the HupA titers during fermentation by adding Huperzia serrata extracts (HSE), L-lysine, and acetic acid into the culture as inducers. Adding trace amounts of HupA clearly improved the HupA production of Shiraia sp. Slf14, reaching a maximum content of approximately 40μgg(-1). Moreover, the addition of HSE and L-lysine promoted HupA production in the flask fermentation. The aforementioned bioprocessing strategy may be potentially applied to other endophytic fungal culture systems for the efficient production of plant secondary metabolites.

Indentification of huperzine A-producing endophytic fungi isolated from Huperzia serrata

This present study was designed to investigate the production of huperzine A (HupA), an acetylcholine inhibitor, which was produced by an endophytic fungi isolated from Huperzia serrata. Screening of 94 endophytic fungal isolates obtained from plant H. serrata was carried out for the production of HupA. Their morphological characteristics were studied and rDNA sequence analysis was carried out. The cultures were grown in liquid culture medium and the extracted metabolites were analyzed by thin layer chromatography and high performance liquid chromatograph for the presence of HupA. The DPPH scavenging ratio and inhibition ratio of acetylcholinesterase (AchE) of the same were determined. 3 out of 94 strains i.e. S29, L44 and S94 showed significant AchE-inhibitory activity and antioxidant activity. Strain L44 which exhibited maximum yield of HupA (37.63μg/g on dry weight basis) was identified as Trichoderma species by ITS sequence analysis. In conclusion, endophytic fungi from H. serrata can be used as a new resource of HupA.

Ethanol and Methanol Can Improve Huperzine A Production from Endophytic Colletotrichum gloeosporioides ES026

Huperzine A (HupA) is a plant alkaloid that is of great interest as a therapeutic candidate for the treatment of Alzheimer's disease. However, the current production of HupA from plants in large quantity is unsustainable because the plant resource is scarce and the content of HupA in plants is extremely low. Surprisingly, this compound was recently found to be produced by various endophytic fungi, which are much more controllable than the plants due to simpler genetics and ease of manipulation. However, it might be due to the innate properties of endophytic symbiosis, that production of this chemical in large quantity from endophytes has not yet been put into practice. Endophytic Colletotrichum gloeosporioides ES026 was previously isolated from a HupA producing plant and the fungi also proved to produce HupA. In this study, various fermentation conditions were tried to optimize the production of HupA from C. gloeosporioides ES026. Optimization of these parameters resulted in a 25.58% increase in HupA yield. Potato extracts supplemented with glucose or sucrose but not maltose facilitated HupA producing from the fungi. A final concentration of 0.5–2% ethanol stimulated the growth of fungi while methanol with the same treatment slightly inhibited the growth. However, both methanol and ethanol greatly increased the HupA production with the highest yield of HupA (51.89% increment) coming from ethanol treatment. Further analysis showed that both ethanol and methanol were strong inducers of HupA production, while ethanol was partially used as a carbon source during fermentation. It was noticed that the color of that ethanol treated mycelia gradually became dark while methanol treated ones stayed grey during fermentation. The present study sheds light on the importance of optimizing the fermentation process, which, combined with effective inducers, maximizes production of chemicals of important economic interest from endophytic fungi.

Production of huperzine A and other Lycopodium alkaloids in Huperzia species grown under controlled conditions and in vitro

A UPLC-MS method was developed for quantifying huperzine A (HupA), an anti-Alzheimer's disease (AD) drug candidate from the traditional Chinese medicine Qian Ceng Ta (Huperzia serrata), in samples of 11 Huperzia genus plants. The highest content of HupA was found in Huperzia pinifolia. The accumulation of various Lycopodium alkaloids was monitored in these tissues using high resolution Q-IMS-TOFMS analysis. Tissue culture of various Huperzia species has been achieved and production of HupA has been confirmed in the callus of H. pinifolia. Furthermore, it was established that the major alkaloid produced by the naturally grown plant and the callus of H. pinifolia changed dramatically from HupA to nankakurine B.

Modification and Validation of a High-Performance Liquid Chromatography Method for Quantification of Huperzine A in Huperzia Crispata

The present study describes a rapid and sensitive HPLC method for the quantification of huperzine A (HupA) in Huperzia crispata (Huperziaceae). The sample extraction and preparation involved a simple, time-saving, single-solvent extraction, with each sample being analyzed within 12 min. The mobile phase was ammonium acetate (0.1 M, pH 6.0)--methanol (64 + 36, v/v) at a flow rate of 1.0 mL/min. Detection was at 308 nm. The calibration curve was linear from 0.049 to 7.84 microg (R2 = 0.9997), with intraday and interday precision RSD of less than 2%. The extraction recovery rate was over 98.49%. Quantification of HupA was performed using this modified method, and the content of HupA was 1.86 times higher in the whole plant of H. crispata (218.17 +/- 1.55 microg/g) than in that of H. serrata (117.03 +/- 2.97 microg/g). In the whole plant of H. crispata, HupA mainly accumulated in the actively growing shoot tips, the apical bud, and the 10 youngest leaves, reaching 455.23 +/- 2.97 microg/g. The content of HupA in the samples from sunshine-sheltered sites was 3.45 times higher than in that from sunshine-abundant sites. The satisfactory results indicate that this modified method can be applied in the quality control of large-scale Huperziaceae plant extracts and that changes should be made in the cultivation of H. crispata so as to maximize the production of HupA.

A novel endophytic Huperzine A-producing fungus, Shiraia sp. Slf14, isolated from Huperzia serrata

To characterize and identify a novel Huperzine A (HupA)-producing fungal strain Slf14 isolated from Huperzia serrata (Thunb. ex Murray) Trev. in China. 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, (1) H 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. 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. 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.