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.

Molecular Evolution and Functional Characterization of a Bifunctional Decarboxylase Involved in Lycopodium Alkaloid Biosynthesis.

Lycopodium alkaloids (LAs) are derived from lysine (Lys) and are found mainly in Huperziaceae and Lycopodiaceae. LAs are potentially useful against Alzheimer's disease, schizophrenia, and myasthenia gravis. Here, we cloned the bifunctional lysine/ornithine decarboxylase (L/ODC), the first gene involved in LA biosynthesis, from the LA-producing plants Lycopodium clavatum and Huperzia serrata We describe the in vitro and in vivo functional characterization of the L. clavatum L/ODC (LcL/ODC). The recombinant LcL/ODC preferentially catalyzed the decarboxylation of l-Lys over l-ornithine (l-Orn) by about 5 times. Transient expression of LcL/ODC fused with the amino or carboxyl terminus of green fluorescent protein, in onion (Allium cepa) epidermal cells and Nicotiana benthamiana leaves, showed LcL/ODC localization in the cytosol. Transgenic tobacco (Nicotiana tabacum) hairy roots and Arabidopsis (Arabidopsis thaliana) plants expressing LcL/ODC enhanced the production of a Lys-derived alkaloid, anabasine, and cadaverine, respectively, thus, confirming the function of LcL/ODC in plants. In addition, we present an example of the convergent evolution of plant Lys decarboxylase that resulted in the production of Lys-derived alkaloids in Leguminosae (legumes) and Lycopodiaceae (clubmosses). This convergent evolution event probably occurred via the promiscuous functions of the ancestral Orn decarboxylase, which is an enzyme involved in the primary metabolism of polyamine. The positive selection sites were detected by statistical analyses using phylogenetic trees and were confirmed by site-directed mutagenesis, suggesting the importance of those sites in granting the promiscuous function to Lys decarboxylase while retaining the ancestral Orn decarboxylase function. This study contributes to a better understanding of LA biosynthesis and the molecular evolution of plant Lys decarboxylase.

[Cloning and analysis of squalene synthase (HsSQS1) gene in Huperzia serrata].

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.

[Cloning and bioinformatics analysis of cycloartenol synthase (HcCAS1) gene in Huperzia carinata].

OBJECTIVE: To clone and sequence the open reading frame of cycloartenol synthase (CAS) from Huperzia carinata. METHOD: After 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. RESULT: One 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. CONCLUSION: This 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.

[Cloning, expression and functional identification of a type III polyketide synthase gene from Huperzia serrata].

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).

[Phylogeny relationship and molecular identification of ten Huperzia species (Huperziaceae) based on matK gene sequences].

OBJECTIVE: To study the phylogeny relationship and molecular identification of 10 species from Huperzia (Huperziaceae) based on matK gene sequences data. METHOD: Total DNA of nine species from Huperzia was extracted; matK gene sequence was amplified by PCR. PCR product was directly sequenced after purification. RESULT: The chloroplast matK gene nucleotide sequences from 9 species of Huperzia species were sequenced. The matK gene nucleotide sequences length was 1 589 bp. Analysis with Huperzia lucidula matK gene nucleotide sequences (download from GenBank) and taking Lycopodiella cernua as outgroup, Maximum Parsimony, Neighbor-Joining analyses and genetic distances were conducted using MEGA 3.1 software. 35 variable sites and 35 parsimony informative sites have been found. Pairwise genetic distances among 10 species of Huperzia was 1.59% - 0.25%. CONCLUSION: The results were consistent with the taxonomy in morphological of Huperzia. But H. longipetiolata and H. serrata were resolved into in different clade. There are 19 different sites of matK gene sequences between H. longipetiolata and H. serrata, the genetic distances is 0.121%. It is suggested H. longipetiolata should be as an independent species.