蛇足石杉中赖氨酸脱羧酶基因的克隆表达及功能鉴定

Gene cloning and functional characterization of a lysine decarboxylase from Huperzia serrata

赖氨酸脱羧酶是石杉碱甲等石松生物碱上游生物合成步骤中催化赖氨酸脱羧生成1,5-戊二胺(尸胺)的关键酶。通过对蛇足石杉(Huperzia serrata)转录组数据的挖掘分析,并结合RT-PCR技术从蛇足石杉中成功克隆出3个赖氨酸脱羧酶基因(HsLDC-L1、HsLDC-L2、HsLDC-L3);利用在线生物信息学分析软件以及DNAMAN、MEGA 7.0等对上述3个基因编码蛋白的性质、二级和三级结构、序列同源性、进化关系等进行了预测分析,发现3个基因编码蛋白均具有植物赖氨酸脱羧酶的保守结构域和活性位点,且与文献报道的产石杉碱甲植物中的赖氨酸脱羧酶具有较近的亲缘关系。进一步尝试利用不同载体在大肠杆菌中对上述3个基因进行表达,结果仅HsLDC-L1与载体pCold TF构建的重组质粒在大肠杆菌E.coli BL21(DE3)中成功实现可溶性表达,利用Ni^(2+)亲和色谱柱分离纯化得到重组蛋白HsLDC-L1;HsLDC-L1全长由469个氨基酸组成,理论分子质量为50.50 kDa。体外酶活性分析证明HsLDC-L1可以催化赖氨酸脱羧生成尸胺。底物适应性实验发现HsLDC-L1还可以催化鸟氨酸脱羧生成腐胺,但不能催化酪氨酸、苯丙氨酸、色氨酸、组氨酸脱羧。上述结果为进一步研究包括石杉碱甲在内的石松类生物碱的生物合成提供参考,为利用合成生物学策略构建产Δ^(1)-哌啶、石榴碱等石松类生物碱生物合成关键前体的工程菌提供重要的基因元件。

Lysine decarboxylase is a key enzyme involved in the upstream biosynthesis of lycopodium alkaloids(LAs)such as huperzine A,contributing to the decarboxylation of lysine to 1,5-pentanediamine(cadaverine).Three lysine decarboxylase genes(HsLDC-L1,HsLDC-L2,HsLDC-L3)were successfully cloned from Huperzia serrata using transcriptomic sequence data mining strategy combined with reverse transcription PCR.The physicochemical properties,secondary and tertiary structures,amino acid identities,and evolutionary relationship of the three LDCs were analyzed by online bioinformatics analysis platforms and DNAMAN,MEGA 7.0 software,revealing that all of these proteins had the conserved PLP binding domain and active site residues were completely conserved in LDCs.Phylogenetic analysis showed that these LDCs were located in the same branch as other known LDCs from LA-producing plants.Accordingly,the ORFs of these three HsLDCs were inserted into different expression plasmids for further expression in E.coli.However,only HsLDC-L1 was successfully expressed in E.coli BL21(DE3)by inserting into a pCold TF vector.The recombinant protein was purified by Ni^(2+)affinity chromatography purification.HsLDC-L1 contains 469 amino acid residues,with a calculated molecular weight of 50.50 kDa.HsLDC-L1expectedly catalyzed the decarboxylation of lysine to produce cadaverine.In addition,HsLDC-L1 can also catalyze the generation of putrescine from ornithine.However,it cannot catalyze the decarboxylation of tyrosine,phenylalanine,tryptophan and histidine.The results not only provide insight into the biosynthesis of LAs including huperzine A,but also provide a critical genetic element for the overproduction of Δ^(1)-piperideine and pelletierine,the essential biosynthetic precursors of LAs,using synthetic biology strategies.