Trehalose promotes high-level heterologous expression of 4,6-α-glucanotransferase GtfR2 in Escherichia coli and mechanistic analysis.
Trehalose promotes high-level heterologous expression of 4,6-α-glucanotransferase GtfR2 in Escherichia coli and mechanistic analysis.
复制标题
DOI：
10.1016/j.ijbiomac.2022.05.037
复制
发表时间：
2022-05
影响因子：
8.2
通讯作者：
D. Rao;Lei Wang;Runtian Huo;Lingqia Su;Zhiyong Guo;Weikang Yang;Beibei Wei;Xiumei Tao;Sheng Chen;Jing Wu
中科院分区：
文献类型：
--
作者：
D. Rao;Lei Wang;Runtian Huo;Lingqia Su;Zhiyong Guo;Weikang Yang;Beibei Wei;Xiumei Tao;Sheng Chen;Jing Wu
4,6-α-Glucanotransferases (4,6-α-GTs) hold great potential for applications in the food and medical industries because of their efficient transglycosylation ability. However, it is relatively difficult to achieve high soluble expression because of their high molecular weight and multidomain nature. In this study, 4,6-α-GT ofBurkholderiasp. (GtfR2) was successfully expressed inE. coli, and the activity attained 1.55 × 104U/mL by traditional fermentation optimization. However, a large number of inactive inclusion bodies of GtfR2 were still present due to aggregation and precipitation. The trehalose-mediated strategy was first proposed and applied in the fermentation process of GtfR2. Trehalose addition significantly reduced inclusion bodies, resulting in an increase in GtfR2 activity (6.48 × 104U/mL), which was 4.20 times higher than that of the control group. Our molecular dynamics simulations revealed that trehalose could spontaneously stabilize the conformational dynamics of GtfR2 by binding to the groove, loop, α-helix and N-terminal unstable regions on the surface. This strategy was also available to enhance the soluble expression of other 4,6/4,3-α-GTs, which were increased by 3.03–77.19 times. This study is the first to observe that trehalose can inhibit the aggregation and precipitation of GtfR2, which provides a new perspective for the recombinant expression of 4,6/4,3-α-GTs.
