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.
Penicillium chrysogenum/genetics* ; Huperzia/microbiology* ; Acrylates ; Multigene Family

A total of 27 endophytic fungal strains were isolated from Huperzia serrata,which were richly distributed in the stems and leaves while less distributed in roots. The 27 strains were identified by Internal Transcribed Spacer( ITS) r DNA molecular method and one of the strains belongs to Basidiomycota phylum,and other 26 stains belong to 26 species,9 general,6 families,5 orders,3 classes of Ascomycota Phylum. The dominant strains were Colletotrichum genus,belonging to Glomerellaceae family,Glomerellales order,Sordariomycetes class,Ascomycota Phylum,with the percentage of 48. 15%. The inhibitory activities of the crude extracts of 27 endophytic fungal strains against acetylcholinesterase( ACh E) and nitric oxide( NO) production were evaluated by Ellman's method and Griess method,respectively. Crude extracts of four fungi exhibited inhibitory activities against ACh E with an IC50 value of 42. 5-62. 4 mg·L~(-1),and some fungi's crude extracts were found to inhibit nitric oxide( NO) production in lipopolysaccharide( LPS)-activated RAW264. 7 macrophage cells with an IC50 value of 2. 2-51. 3 mg·L~(-1),which indicated that these fungi had potential anti-inflammatory activities.The chemical composition of the Et OAc extract of endophytic fungus HS21 was also analyzed by LCMS-IT-TOF. Seventeen compounds including six polyketides,four diphenyl ether derivatives and seven meroterpenoids were putatively identified.
Acetylcholinesterase ; Animals ; Anti-Inflammatory Agents ; isolation & purification ; pharmacology ; Ascomycota ; chemistry ; classification ; isolation & purification ; Cholinesterase Inhibitors ; isolation & purification ; metabolism ; Endophytes ; classification ; isolation & purification ; Huperzia ; microbiology ; Mice ; RAW 264.7 Cells

Huperzine A is a promising drug to treat Alzheimer's disease (AD). To date, its biosynthetic pathway is still unknown. Lysine decarboxylase (LDC) has been proposed to catalyze the first-step of the biosynthesis of huperzine A. To identify and characterize LDCs from Huperzia serrata, we isolated two LDC fragments (LDC1 and LDC2) from leaves of H. serrata by RT-PCR and then cloned them into pMD 19-T vector. Sequence analysis showed that LDC1 and LDC2 genes shared 95.3% identity and encoded the protein of 212 and 202 amino acid residues respectively. Thus, we ligated LDC genes into pET-32a(+) to obtain recombinant expressing vectors pET-32a(+)/LDC1 and pET-32a(+)/LDC2 respectively. We further introduced two expression vectors into Escherichia coli BL21(DE3) and cultured positive colonies of E. coli in liquid LB medium. After inducing for 4 hours with 260 μg/mL IPTG at 30 degrees C, soluble recombinant Trx-LDC1 and Trx-LDC2 were obtained and isolated for purification using a Ni-NTA affinity chromatography. We incubated purified recombinant proteins with L-lysine in the enzyme reaction buffer at 37 degrees C and then derived the reaction products using dansyl chloride. It was found that both Trx-LDC1 and Trx-LDC2 had decarboxylase activity, could convert L-lysine into cadaverine by way of thin layer chromatography assay. Further, bioinformatics analysis indicated that deduced LDC1 and LDC2 had different physicochemical properties, but similar secondary and three-dimensional structures.
Carboxy-Lyases ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; metabolism ; Genetic Vectors ; Huperzia ; enzymology ; genetics ; Lysine ; metabolism ; Plant Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

AIM: To investigate the chemical constituents from the whole plants of Phlegmariurus fargesii. METHOD: Compounds were isolated by repeated silica gel column chromatography. Their structures were elucidated by spectroscopic methods and chemical correlation. The acetylcholinesterase (AChE) inhibitory activity of the isolated compounds was evaluated. RESULTS: A new Lycopodium alkaloid, lycopodine N-oxide (1), along with lycopodine (2), 8,15-dehydrolycopodine (3), 6α-hydroxylycopodine (4), deacetyllycoclavine (5), N-methylhuperzine B (6), lycodine (7), and phlegmarine (8), was isolated. CONCLUSION Compound 1 is a new Lycopodium alkaloid, and compound 3 was obtained from nature for the first time. Other alkaloids are isolated from this plant for the first time.
Alkaloids ; chemistry ; isolation & purification ; Huperzia ; chemistry ; Molecular Structure ; Plant Extracts ; chemistry ; isolation & purification ; Quinolizines ; chemistry ; isolation & purification

OBJECTIVETo clone and sequence the open reading frame of cycloartenol synthase (CAS) from Huperzia carinata.

METHODAfter searching the transcriptome dataset of H. carinata, one unique sequence containing oxide squalene cyclases domain was discovered. The primers were designed according to the cDNA sequence of CAS from the dataset. And then, the open reading frame of CAS was cloned by RT-PCR strategy with the template of mixed RNA extracted from root, stem and leaf of H. carinata. The bioinformatic analysis of this gene and its corresponding protein was performed.

RESULTOne unique sequence of CAS, named as HcCAS1 (GenBank accession number JN790125) , was cloned from H. carinata. The open reading frame of HcCAS1 consists of 2 474 bp, encoding one polypeptide with 757 amino acids.

CONCLUSIONThis study cloned and analyzed CAS from H. carinata for the first time. The result will provide a foundation for exploring the mechanism of sterol biosynthesis in Huperziaceae plants.

Amino Acid Sequence ; Cloning, Molecular ; Computational Biology ; Evolution, Molecular ; Huperzia ; enzymology ; genetics ; Intramolecular Transferases ; chemistry ; genetics ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Phylogeny ; Protein Structure, Secondary

OBJECTIVETo screen out fungus strains with acetylcholinesterase inhibitory activity from Huperzia serrata.

METHODEndophytic fungi fermentation products from 59 H. serrata strains were stained with acetylcholinesterase hydrolyzed alpha-naphthaleneacetic ethyl ester and fast blue B salt, and screened for acetylcholinesterase inhibitory activity with thin-layer chromatography-bioautography. Target strains were classified and identified through the sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics.

RESULTFungus strain LQ2F01 from H. serrata showed positive color reaction in the screening for acetylcholinesterase inhibitory activity. The sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics showed the strain LQ2F01 belonged to Acremonium.

CONCLUSIONEndophytic Fungi LQ2F01 from H. serrata shows identical acetylcholinesterase inhibitory activity with the host plant, which is of great significance to the development of natural medicines and the studies on the relationship between the endophytic gungi and the host plant.

Acetylcholinesterase ; metabolism ; Acremonium ; genetics ; metabolism ; Cholinesterase Inhibitors ; isolation & purification ; metabolism ; Chromatography, Thin Layer ; DNA, Fungal ; chemistry ; genetics ; DNA, Ribosomal ; chemistry ; genetics ; Diazonium Compounds ; metabolism ; Fungi ; classification ; genetics ; metabolism ; Huperzia ; microbiology ; Hydrolysis ; Naphthaleneacetic Acids ; metabolism ; Phylogeny ; RNA, Ribosomal, 18S ; classification ; genetics ; RNA, Ribosomal, 5.8S ; classification ; genetics ; Sequence Analysis, DNA

Squalene synthase (SQS) is a key enzyme in plant terpenoid biosynthetic pathway. This study focused on cloning and analysis of Huperzia serrata SQS (HsSQS1) gene. After searching the transcriptome dataset of H serrata, one unique sequence encoding SQS was discovered. The primers were designed according to the transcript sequence of HsSQS1 from the H. serrata transcriptome dataset. The open reading frame of HsSQS1 was cloned using RT-PCR strategy. The bioinformatic analysis of this gene and its corresponding protein were performed. The cDNA (named as HsSQS1) contains a 1263 bp open reading frame and encodes a predicted protein of 420 amino acids. The GenBank accession number for this gene is JQ004938. HsSQS1 contains two transmembrane regions, without signal peptide. The conserved domain of squalene synthase was presented in HsSQS1. HsSQS1 was more abundant in H. serrata root than in leaf and stem. This study cloned and analyzed squalene synthase gene from H. serrata for the first time. The result will provide a foundation for exploring the mechanism ofterpenoid biosynthesis in H. serrata plants.
Amino Acid Sequence ; Biosynthetic Pathways ; Cloning, Molecular ; DNA, Complementary ; genetics ; Expressed Sequence Tags ; Farnesyl-Diphosphate Farnesyltransferase ; genetics ; isolation & purification ; metabolism ; Genes, Plant ; genetics ; Huperzia ; enzymology ; genetics ; Molecular Sequence Data ; Open Reading Frames ; Phylogeny ; Plant Leaves ; enzymology ; Plant Roots ; enzymology ; Plant Stems ; enzymology ; Plants, Medicinal ; enzymology ; genetics ; Triterpenes ; chemistry

A total of 127 strains of endophytic fungi were isolated from roots, branches and leaves of Huperzia serrata. These strains were identified into 19 genera based on morphological characters and ribosomal DNA (rDNA) sequence analysis, there into Penicillium, Aspergillus and Podospora were dominant populations in H. serrata. From analysis results we found some endophytic fungi showed a certain degree of tissue preference. The isolation rate and colonization rate of stems were both larger than those of leaf and roots. After testing the acetylcholinesterase (AChE) inhibitory activity of these endophytic fungi, a total of 39 endophytic fungi belonging to 15 genera showed AChE inhibition. Eleven endophytic fungi showed potent AChE inhibition, 7 of which were isolated from leaf. The research not only provided theoretical basis for developing and utilizing the resources of endophytic fungi in H. serrata but also showed a new path for searching medicines resource which has AChE inhibitory activity.
Cholinesterase Inhibitors ; pharmacology ; Fungi ; classification ; isolation & purification ; Huperzia ; microbiology

A cDNA encoding novel type III polyketide synthase (PKS) was cloned and sequenced from young leaves of Chinese club moss Huperzia serrata (Thunb.) Trev. by RT-PCR using degenerated primers based on the conserved sequences of known CHSs, and named as H. serrata PKS2. The terminal sequences of cDNA were obtained by the 3'- and 5'-RACE method. The full-length cDNA of H. serrata PKS2 contained a 1212 bp open reading frame encoding a 46.4 kDa protein with 404 amino acids. The deduced amino acid sequence of H. serrata PKS2 showed 50%-66% identities to those of other chalcone synthase super family enzymes of plant origin. The recombinant H. serrata PKS2 was functionally expressed in Escherichia coli with an additional hexahistidine tag at the N-terminus and showed unusually versatile catalytic potency to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, the enzyme accepted bulky starter substrates N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 1, 3-dihydroxy-N-methylacridone. Interestingly, H. serrata PKS2 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).
Acyltransferases ; genetics ; isolation & purification ; metabolism ; Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Plant ; genetics ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Huperzia ; enzymology ; genetics ; Molecular Sequence Data ; Plant Leaves ; enzymology ; genetics ; Plants, Medicinal ; enzymology ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Sequence Alignment ; Substrate Specificity

A precise and selective reversed phase high-performance liquid chromatographic method (HPLC) was used to quantify the levels of huperzine A in samples of three Huperzia serrata populations with a total of 73 individuals located in Zhejiang, Guangxi, Chongqing, respectively, as well as in one-to-one samples of these 73 individuals introduced in same site after one year. Huperzine A content variation both among and within populations, and the dynamic change of this alkaloid occurring in same population after one year introduction, were analyzed using SPSS 13.0 software (Coefficient of variation, One-way ANOVA analysis, Paired-samples T tests). The results indicated that huperzine A content varied significantly by geographical locations, especially change with longitude, i. e., the order of the huperzine A content was CQ population > GX population > ZJ population. The coefficients of variation (CV) were as follows: 0.36 (CQ), 0.44 (GX) and 0.40 (ZJ). This indicated that there was plentiful diversity concerned with huperzine A content among individuals within population. Moreover, this high diversity was still maintained after one year introduction. ANOVA analysis showed that there was significant difference among populations in huperzine A content. Finally, the significant change of huperzine A content was not observed in all three populations after one year introduction. The results presented in this study could provide evidence that the huperzine A content variation of H. serrata is the results of an interaction between genes and the environment, by comparison, is mainly controlled by genetic factor.
Chromatography, High Pressure Liquid ; methods ; Chromatography, Reverse-Phase ; methods ; Drugs, Chinese Herbal ; Huperzia ; chemistry