Yamaguchi Riko, Akter Shamoli, Kanehama Aki, Iwamoto Takahiro, Hasegawa Meme, Ito Akeno, Nishimukai Megumi, Yamada Miwa, Kashiwagi Akiko
Faculty of Agriculture and Life Science, Hirosaki University, Bunkyo-cho 3, Hirosaki, Aomori 036-8561, Japan.
The United Graduate School of Agricultural Science, Iwate University, Ueda-3, Morioka, Iwate 020-8550, Japan.
Lett Appl Microbiol. 2023 Apr 3;76(4). doi: 10.1093/lambio/ovad049.
Plasmalogens are a subclass of glycerophospholipids that have a vinyl-ether bond at the sn-1 position and are thought to have several physiological functions. The creation of non-natural plasmalogens with functional groups is desired for the establishment of the prevention of diseases caused by the depletion of plasmalogens. Phospholipase D (PLD) has both hydrolysis and transphosphatidylation activities. In particular, PLD from Streptomyces antibioticus has been investigated extensively due to its high transphosphatidylation activity. However, it has been difficult to stably express recombinant PLD in Escherichia coli and to express it as a soluble protein. In this study, we used the E. coli strain, SoluBL21™, and achieved stable PLD expression from the T7 promoter and increased soluble fraction in the cell. We also improved the purification method of PLD using His-tag at the C terminus. We obtained PLD with ∼730 mU mg-1 protein of specific activity, and the yield was ∼420 mU l-1 culture, corresponding to 76 mU per gram of wet cells. Finally, we synthesized a non-natural plasmalogen with 1,4-cyclohexanediol bound to the phosphate group at the sn-3 position by transphosphatidylation of the purified PLD. This method will contribute to the expansion of the chemical structure library of non-natural plasmalogens.
缩醛磷脂是甘油磷脂的一个亚类，其在sn-1位具有乙烯基醚键，被认为具有多种生理功能。为了预防因缩醛磷脂缺乏引起的疾病，人们期望合成带有官能团的非天然缩醛磷脂。磷脂酶D（PLD）具有水解和转磷脂酰基活性。特别是来自抗生链霉菌的PLD，因其高转磷脂酰基活性而受到广泛研究。然而，在大肠杆菌中稳定表达重组PLD并将其表达为可溶性蛋白一直很困难。在本研究中，我们使用了大肠杆菌菌株SoluBL21™，实现了从T7启动子稳定表达PLD，并增加了细胞中的可溶性部分。我们还改进了使用C端His标签的PLD纯化方法。我们获得了比活性约为730 mU mg-1蛋白的PLD，产量约为420 mU l-1培养物，相当于每克湿细胞76 mU。最后，我们通过纯化的PLD的转磷脂酰基化反应，合成了一种在sn-3位的磷酸基团上结合有1,4-环己二醇的非天然缩醛磷脂。该方法将有助于扩展非天然缩醛磷脂的化学结构库。
