Molecular Cloning And Expression Of Lysine Decarboxylase And Copper Amine Oxidase Gene Of Endophytic Fungus Shiraia Sp. Slf14 From Huperzia Serrata

Huperzine A(HupA) is a kind of alkaloids and applied in the clinical on Alzheimer's disease(AD). HupA was mainly extracted from Lycopodium such as H. serrata, that resulted in the overexploitation of plants and environmental disruption. The HupA-producing endophytic fungi can be used in microbial fermentation of HupA. Illumination of biosynthetic pathway was usually based on genomics, metabolic engineering and combinatorial biosynthesis technologies, however, the biosynthetic pathway of HupA was unclear. In this study, an endophytic fungus Shiraia sp. Slf14 is capable of producing HupA, which was isolated from H. serrata. Shiraia sp. Slf14 has been genomic sequencing, and the genome information have been got. The Lysine decarboxylase(LDC) gene and copper amine oxidase(CAO) gene of Shiraia sp. Slf14 was revealed by bioinformatics analysis, which were involved in the first and second step of HupA biosynthesis, respectively. Thus, it will be of significant interest to research the LDC and CAO genes and identify their function.Based on the complete genome sequence of Shiraia sp. Slf14, the LDC gene of Shiraia sp. Slf14 was predicted. In this study, RT-PCR was performed with cDNA as the template to amplify the LDC fragment. A 687 bp DNA fragment was obtained from Shiraia sp. Slf14. The nucleotide sequence has been submitted to the Gen Bank database(GenBank ID: KT362170). Subsequently, this DNA fragment was subcloned into the pET-22b(+) vector and pET-32a(+) vector respectively, to yield the expression plasmid pET-22b-LDC and pET-32a-LDC. The recombinant LDC and Trx-LDC protein were expressed in E. coli BL21(DE3) and purified by Ni-chelating affinity column chromatography. The purified LDC and Trx-LDC protein with a molecular weight of 24 kDa and 42 kDa, respectively. The sizes correspond to the predicted sizes. The reaction mixture was prepared with the purified LDC and TrxLDC protein, respectively. Their catalytic activities were measured by Thin Layer Chromatography(TLC). TLC analysis revealed that LDC and Trx-LDC were both having catalytic abilities. The physicochemical properties and structures of the LDC was obtained by bioinformatics softwares. The predicted secondary structure of LDC showed that ?-helix had 37.3 %, ?-strand occupied 11.4 %, coil occupied 11.4 %. According to predicted tertiary structure, this LDC protein was dimer, and N-terminal was exposed outsides while C-terminal was used to form dimer structure. Based on amino acid sequence of fungi and plants LDC, phylogenetic tree analysis revealed th at LDC of Shiraia sp. Slf14 is closely related to Aspergillus clavatus NRRL 1 LDC, but far from Huperzia serrata LDCs.Based on the complete genome sequence of Shiraia sp. Slf14 and conserved sequences of the known CAOs, the SsCAO gene of Shiraia sp. Slf14 was predicted. The 2031 bp DNA fragment was obtained from Shiraia sp. Slf14 by RT-PCR, which encodes a copper amine oxidase. The nucleotide sequence has been submitted to the GenBank database(Gen Bank ID: KT362171). Subsequently, this DNA fragment was sub-cloned into the pET22b(+) vector to yield the expression plasmid pET-SsCAO, successfully. The recombinant SsCAO protein was expressed in E. coli BL21(DE3) and purified by Ni-chelating affinity column chromatography. The purified SsCAO protein migrated as a single band with a molecular weight of 76 kDa. The size of CAO protein corresponds to the predicted size of SsCAO(75,985 Da). The reaction mixture was prepared with the purified SsCAO protein. GC–MS analysis revealed that recombinant CAO can accept cadaverine as substrate to produce 5-aminopentanal. In contrast, when the boiled inactive CAO was added to the enzyme reaction mixture, the catalytic activity was undetectable. The physicochemical properties and structures of SsCAO was obtained by bioinformatics softwares. The predicted structure of SsCAO showed that ?-helixes were concentrated at N/C-terminal, and ?-strand occupied a big proportion. Analysis of the amino acid sequence of the SsCAO protein revealed that it contains the Asp catalytic base( Tyr299, Lys309 and Asp313), conserved motif Asn-Tyr-Asp/Glu, and three copper-binding histidines(His447 ?His449?His607). Phylogenetic tree analysis indicated that CAO from Shiraia sp. Slf14 is closely related to Stemphylium lycopersici CAO, far from H. serrata CAO or other plant CAOs. These results suggested that the SsCAO gene in Shiraia sp. Slf14 is not a result from horizontal gene transfer from H. serrata. Characterization of LDC and SsCAO from Shiraia sp. Slf14 not only provide information for further understanding of the HupA biosynthetic pathway, but also paves a way for future biosynthesis of HupA.