Zhai Yafei, Han Donglei, Pan Ying, Wang Shuaishuai, Fang Junqiang, Wang Peng, Liu Xian-wei
National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250100, People's Republic of China; The State Key Laboratory of Microbial Technology and School of Life Science, Shandong University, Jinan, Shandong 250100, People's Republic of China.
The State Key Laboratory of Microbial Technology and School of Life Science, Shandong University, Jinan, Shandong 250100, People's Republic of China.
Enzyme Microb Technol. 2015 Feb;69:38-45. doi: 10.1016/j.enzmictec.2014.12.001. Epub 2014 Dec 9.
Guanosine 5'-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5'-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue.
5'-二磷酸鸟苷（GDP）-岩藻糖是岩藻糖基转移酶催化合成含岩藻糖生物分子所必需的供体底物，这些生物分子已被发现参与多种生物学功能。在本研究中，将脆弱拟杆菌中GDP-岩藻糖生物合成的补救途径引入大肠杆菌。此外，通过过表达参与GTP生物合成补救途径的酶，增强了GDP-岩藻糖生物合成的必需底物三磷酸鸟苷（GTP）的生物合成。比较了携带不同重组酶组合的代谢工程菌株的生产能力。表达Fkp、Gpt、Gmk和Ndk的菌株摇瓶发酵获得的最大GDP-岩藻糖含量为4.6±0.22μmol/g（干细胞质量），是仅表达Fkp的菌株的4.2倍。通过补料分批发酵，GDP-岩藻糖含量进一步升至6.6±0.14μmol/g（干细胞质量）。除了比以前基于GDP-岩藻糖生物合成从头途径的发酵过程具有更高的生产率外，本研究中建立的方案还具有成为生产对岩藻糖残基进行化学修饰的GDP-岩藻糖类似物的潜在途径的优势。
