蛇足石杉内生真菌Shiraia sp. Slf14中赖氨酸脱羧酶基因的克隆、表达及功能

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

摘要:

[目的] 阿尔茨海默症治疗药物石杉碱甲(Huperzine A, HupA)的生物合成途径起始于赖氨酸脱羧酶(Lysine decarboxylase, LDC)。本研究克隆及表达了来源于产HupA的植物内生真菌的LDC基因,并研究了其功能。[方法] 采用RT-PCR扩增法,从一株产HupA的蛇足石杉内生真菌Shiraia sp. Slf14获得LDC基因,构建表达质粒pET-22b-LDC与pET-32a-LDC,转化感受态细胞E. coli BL21,加入IPTG至终浓度为1×10^-3 mol/L,于24℃、200 r/min培养8 h,诱导表达LDC蛋白质;通过Ni²⁺金属亲和层析纯化重组LDC并建立酶促反应体系,利用TLC检测了LDC催化活性。利用生物信息学软件分析了LDC的理化性质及蛋白质的空间结构。[结果] 成功克隆并异源表达出重组蛋白LDC与Trx-LDC,经SDS-PAGE电泳鉴定分子量分别为24.4 kDa和42.7 kDa,与预计大小相符。TLC结果表明LDC与Trx-LDC均具有赖氨酸脱羧酶活性。[结论] 本研究从产HupA的蛇足石杉内生真菌Shiraia sp. Slf14中成功克隆到LDC基因并进行了异源表达,检测到了其催化活性,为丰富LDC分子信息及阐明内生真菌中HupA生物合成机制提供参考数据。

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.