Molecular cloning, expression and characterization of lysine decarboxylase gene of endophytic fungus Shiraia sp. Slf14 from Huperzia serrata

Abstract:

[Objective] Huperzine A (HupA) was approved as a drug for the treatment of Alzheimer's disease. The HupA biosynthetic pathway was started from lysine decarboxylase (LDC), which catalyzes lysine to cadaverine. In this study, we cloned and expressed an LDC gene from a HupA-producing endophytic fungus, and tested LDC activities. [Methods] An endophytic fungus Shiraia sp. Slf14 from Huperzia serrata was used. LDC gene was obtained by RT-PCR, and cloned into pET-22b(+) and pET-32a(+) vectors to construct recombinant plasmids pET-22b-LDC and pET-32a-LDC. These two recombinant plasmids were transformed into E. coli BL21, cultured for 8 h at 24℃, 200 r/min with 1×10^-3 mol/L IPTG into medium to express the LDC proteins, respectively. LDC proteins were purified by Ni²⁺ affinity chromatography. Catalytic activities were measured by Thin Layer Chromatography. At last, the physicochemical properties and structures of these two LDCs were obtained by bioinformatics software. [Results] LDC and Trx-LDC were expressed in E. coli BL21 successfully. SDS-PAGE analysis shows that the molecular weight of LDC and Trx-LDC were 24.4 kDa and 42.7 kDa respectively, which are consistent with bioinformatics analysis. In addition, TLC analysis reveals that both LDC and Trx-LDC had catalytic abilities. [Conclusion] This work can provide fundamental data for enriching LDC molecular information and reveal the HupA biosynthetic pathway in endophytic fungi.

Key words:lysine decarboxylase;endophytic fungus from Huperzia serrata;huperzine A;cloning and expression;enzyme activity