从脑膜脓毒性伊丽莎白菌 sp.F2 中克隆和功能表征香叶基香叶基二磷酸合酶（GGPPS）。
Cloning and functional characterization of the geranylgeranyl diphosphate synthase(GGPPS)from Elizabethkingia meningoseptica sp.F2.
机构信息
Institute of Intelligent Machines, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, PR China; University of Science and Technology of China, Hefei, 230026, PR China.
Institute of Intelligent Machines, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, PR China.
出版信息
Protein Expr Purif. 2022 Jan;189:105986. doi: 10.1016/j.pep.2021.105986. Epub 2021 Sep 29.
To date, there is no functional characterization of EmGGPPS (from Elizabethkingia meningoseptica sp.F2) as enzymes catalyzing GGPP. In this research, maltose-binding protein (MBP), disulfide bond A (DbsA), disulfide bond C (DbsC), and two other small protein tags, GB1 (Protein G B1 domain) and ZZ (Protein A IgG ZZ repeat domain), were used as fusion partners to construct an EmGGPPS fusion expression system. The results indicated that the expression of MBP-EmGGPPS was higher than that of the other four fusion proteins in E. coli BL21 (DE3). Additionally, using EmGGPPS as a catalyst for the production of GGPP was verified using a color complementation assay in Escherichia coli. In parallel with it, the enzyme activity experiment in vitro showed that the EmGGPPS protein could produce GGPP, GPP and FPP. Finally, we successfully demonstrated MK-4 production in engineered E. coli by overexpression of EmGGPPS.
迄今为止，尚没有对来自弗氏伊丽莎白菌（Elizabethkingia meningoseptica sp.F2）的 EmGGPPS 酶作为 GGPP 催化剂进行功能特征描述。在本研究中，使用麦芽糖结合蛋白（MBP）、二硫键 A（DbsA）、二硫键 C（DbsC）以及另外两个小蛋白标签，GB1（蛋白 G B1 结构域）和 ZZ（蛋白 A IgG ZZ 重复结构域）作为融合伴侣构建了 EmGGPPS 融合表达系统。结果表明，MBP-EmGGPPS 在大肠杆菌 BL21（DE3）中的表达高于其他四种融合蛋白。此外，使用 EmGGPPS 作为 GGPP 产生的催化剂在大肠杆菌中通过颜色互补测定进行了验证。与此同时，体外酶活性实验表明，EmGGPPS 蛋白可以产生 GGPP、GPP 和 FPP。最后，通过过表达 EmGGPPS，我们成功地在工程大肠杆菌中实现了 MK-4 的生产。
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