Förster-Fromme Karin, Schneider Sarah, Sprenger Georg A, Albermann Christoph
Institute of Microbiology, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Biotechnol Lett. 2017 Feb;39(2):219-226. doi: 10.1007/s10529-016-2233-x. Epub 2016 Oct 13.
To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes.
The heterologous expression of the recombinant and codon-adapted human GDP-L-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles. SLC35C1 is an antiporter specific for GDP-L-fucose and depending on the concomitant reverse transport of GMP. The recombinant transporter FucT1 exhibited an activity for the transport of H-GDP-L-fucose with a V of 8 pmol/min mg with a K of 4 µM. The functional expression of SLC35C1 in GDP-L-fucose overproducing E. coli led to the export of GDP-L-fucose to the culture supernatant.
The export of GDP-L-fucose by E. coli provides the opportunity for the engineering of a periplasmatic fucosylation reaction in recombinant bacterial cells.
研究核苷酸活化糖从胞质溶胶跨膜转运至内质网或高尔基体的过程，这是真核生物中糖蛋白和糖脂合成的重要步骤。
重组且密码子优化的人类GDP-L-岩藻糖反向转运蛋白基因SLC35C1（编码N端OmpA信号序列）的异源表达导致一种功能性转运蛋白定位于大肠杆菌的细胞质膜。使用翻转膜囊泡研究了体外转运。SLC35C1是一种对GDP-L-岩藻糖特异的反向转运蛋白，依赖于GMP的伴随反向转运。重组转运蛋白FucT1对H-GDP-L-岩藻糖的转运活性为V 8 pmol/min mg，K为4 μM。SLC35C1在过量产生GDP-L-岩藻糖的大肠杆菌中的功能性表达导致GDP-L-岩藻糖分泌到培养上清中。
大肠杆菌分泌GDP-L-岩藻糖为在重组细菌细胞中构建周质岩藻糖基化反应提供了机会。
