Xu Baofu, Lei Lei, Zhu Xiaocen, Zhou Yiqing, Xiao Youli
CAS Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China; University of Chinese Academy of Sciences, Beijing, 100039, China.
CAS Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
Phytochemistry. 2017 Apr;136:23-30. doi: 10.1016/j.phytochem.2016.12.022. Epub 2017 Jan 12.
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 (k/K, 2.11 s mM) 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.
赖氨酸脱羧是石杉碱甲生物合成的第一步。通过简并PCR和cDNA末端快速扩增（RACE）技术，从蛇足石杉中克隆到6个编码赖氨酸脱羧酶（LDC）的cDNA。其中一种蛇足石杉LDC同工型被鉴定为具有赖氨酸脱羧酶功能。在所有已报道的植物LDC中，蛇足石杉LDC对L-赖氨酸表现出最高的催化效率（k/K，2.11 s mM）。此外，蛇足石杉LDC在烟草叶片中的瞬时表达使尸胺含量从零增加到每克干重0.75毫克。此外，还开发了一种便捷可靠的方法来检测这两种催化产物。利用该新方法，在纯化酶的测定以及转基因烟草叶片中，同时检测到了蛇足石杉LDC和蛇足石杉CAO的酶促产物，分别为尸胺和5-氨基戊醛。这项工作不仅为蛇足石杉中石杉碱甲生物合成途径的前两步提供了直接证据，为进一步阐明该途径铺平了道路，还为石杉碱甲的异源工程生产提供了可能。
